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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Silver nanoparticle production by the fungus
<italic>Fusarium oxysporum</italic>: nanoparticle characterisation and
analysis of antifungal activity against pathogenic yeasts</title><author><name sortKey="Ishida, Kelly" sort="Ishida, Kelly" uniqKey="Ishida K" first="Kelly" last="Ishida">Kelly Ishida</name><affiliation><nlm:aff id="aff1">Laboratório de Quimioterapia Antifúngica, Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP,<country>Brasil</country></nlm:aff></affiliation></author><author><name sortKey="Cipriano, Talita Ferreira" sort="Cipriano, Talita Ferreira" uniqKey="Cipriano T" first="Talita Ferreira" last="Cipriano">Talita Ferreira Cipriano</name><affiliation><nlm:aff id="aff2">Laboratório de Biologia Celular de Fungos</nlm:aff></affiliation></author><author><name sortKey="Rocha, Gustavo Miranda" sort="Rocha, Gustavo Miranda" uniqKey="Rocha G" first="Gustavo Miranda" last="Rocha">Gustavo Miranda Rocha</name><affiliation><nlm:aff id="aff3">Laboratório de Física Biológica</nlm:aff></affiliation><affiliation><nlm:aff id="aff4">Instituto Nacional de Metrologia, Padronização e Qualidade Industrial, Duque de Caxias, RJ,<country>Brasil</country></nlm:aff></affiliation></author><author><name sortKey="Weissmuller, Gilberto" sort="Weissmuller, Gilberto" uniqKey="Weissmuller G" first="Gilberto" last="Weissmüller">Gilberto Weissmüller</name><affiliation><nlm:aff id="aff3">Laboratório de Física Biológica</nlm:aff></affiliation></author><author><name sortKey="Gomes, Fabio" sort="Gomes, Fabio" uniqKey="Gomes F" first="Fabio" last="Gomes">Fabio Gomes</name><affiliation><nlm:aff id="aff5">Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ,<country>Brasil</country></nlm:aff></affiliation></author><author><name sortKey="Miranda, Kildare" sort="Miranda, Kildare" uniqKey="Miranda K" first="Kildare" last="Miranda">Kildare Miranda</name><affiliation><nlm:aff id="aff5">Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ,<country>Brasil</country></nlm:aff></affiliation></author><author><name sortKey="Rozental, Sonia" sort="Rozental, Sonia" uniqKey="Rozental S" first="Sonia" last="Rozental">Sonia Rozental</name><affiliation><nlm:aff id="aff2">Laboratório de Biologia Celular de Fungos</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24714966</idno><idno type="pmc">4015259</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015259</idno><idno type="RBID">PMC:4015259</idno><idno type="doi">10.1590/0074-0276130269</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">001064</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Silver nanoparticle production by the fungus
<italic>Fusarium oxysporum</italic>: nanoparticle characterisation and
analysis of antifungal activity against pathogenic yeasts</title><author><name sortKey="Ishida, Kelly" sort="Ishida, Kelly" uniqKey="Ishida K" first="Kelly" last="Ishida">Kelly Ishida</name><affiliation><nlm:aff id="aff1">Laboratório de Quimioterapia Antifúngica, Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP,<country>Brasil</country></nlm:aff></affiliation></author><author><name sortKey="Cipriano, Talita Ferreira" sort="Cipriano, Talita Ferreira" uniqKey="Cipriano T" first="Talita Ferreira" last="Cipriano">Talita Ferreira Cipriano</name><affiliation><nlm:aff id="aff2">Laboratório de Biologia Celular de Fungos</nlm:aff></affiliation></author><author><name sortKey="Rocha, Gustavo Miranda" sort="Rocha, Gustavo Miranda" uniqKey="Rocha G" first="Gustavo Miranda" last="Rocha">Gustavo Miranda Rocha</name><affiliation><nlm:aff id="aff3">Laboratório de Física Biológica</nlm:aff></affiliation><affiliation><nlm:aff id="aff4">Instituto Nacional de Metrologia, Padronização e Qualidade Industrial, Duque de Caxias, RJ,<country>Brasil</country></nlm:aff></affiliation></author><author><name sortKey="Weissmuller, Gilberto" sort="Weissmuller, Gilberto" uniqKey="Weissmuller G" first="Gilberto" last="Weissmüller">Gilberto Weissmüller</name><affiliation><nlm:aff id="aff3">Laboratório de Física Biológica</nlm:aff></affiliation></author><author><name sortKey="Gomes, Fabio" sort="Gomes, Fabio" uniqKey="Gomes F" first="Fabio" last="Gomes">Fabio Gomes</name><affiliation><nlm:aff id="aff5">Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ,<country>Brasil</country></nlm:aff></affiliation></author><author><name sortKey="Miranda, Kildare" sort="Miranda, Kildare" uniqKey="Miranda K" first="Kildare" last="Miranda">Kildare Miranda</name><affiliation><nlm:aff id="aff5">Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ,<country>Brasil</country></nlm:aff></affiliation></author><author><name sortKey="Rozental, Sonia" sort="Rozental, Sonia" uniqKey="Rozental S" first="Sonia" last="Rozental">Sonia Rozental</name><affiliation><nlm:aff id="aff2">Laboratório de Biologia Celular de Fungos</nlm:aff></affiliation></author></analytic><series><title level="j">Memórias do Instituto Oswaldo Cruz</title><idno type="ISSN">0074-0276</idno><idno type="eISSN">1678-8060</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The microbial synthesis of nanoparticles is a green chemistry approach that
combines nanotechnology and microbial biotechnology. The aim of this study was
to obtain silver nanoparticles (SNPs) using aqueous extract from the filamentous
fungus <italic>Fusarium oxysporum</italic> as an alternative to chemical
procedures and to evaluate its antifungal activity. SNPs production increased in
a concentration-dependent way up to 1 mM silver nitrate until 30 days of
reaction. Monodispersed and spherical SNPs were predominantly produced. After 60
days, it was possible to observe degenerated SNPs with in additional needle
morphology. The SNPs showed a high antifungal activity against
<italic>Candida</italic> and <italic>Cryptococcus</italic> , with minimum
inhibitory concentration values ≤ 1.68 µg/mL for both genera. Morphological
alterations of <italic>Cryptococcus neoformans</italic> treated with SNPs were
observed such as disruption of the cell wall and cytoplasmic membrane and lost
of the cytoplasm content. This work revealed that SNPs can be easily produced by
<italic>F. oxysporum</italic> aqueous extracts and may be a feasible,
low-cost, environmentally friendly method for generating stable and uniformly
sized SNPs. Finally, we have demonstrated that these SNPs are active against
pathogenic fungi, such as <italic>Candida</italic> and
<italic>Cryptococcus</italic> .</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Ahamed, M" uniqKey="Ahamed M">M Ahamed</name></author><author><name sortKey="Alsalhi, Ms" uniqKey="Alsalhi M">MS AlSalhi</name></author><author><name sortKey="Siddiqui, Mkj" uniqKey="Siddiqui M">MKJ Siddiqui</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ahmad, A" uniqKey="Ahmad A">A Ahmad</name></author><author><name sortKey="Mukherjee, P" uniqKey="Mukherjee P">P Mukherjee</name></author><author><name sortKey="Senapati, S" uniqKey="Senapati S">S Senapati</name></author><author><name sortKey="Mandal, D" uniqKey="Mandal D">D Mandal</name></author><author><name sortKey="Khan, Mi" uniqKey="Khan M">MI Khan</name></author><author><name sortKey="Kumar, R" uniqKey="Kumar R">R Kumar</name></author><author><name sortKey="Sastry, M" uniqKey="Sastry M">M 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Mem Inst Oswaldo Cruz</journal-id><journal-id journal-id-type="iso-abbrev">Mem. Inst. Oswaldo Cruz</journal-id><journal-title-group><journal-title>Memórias do Instituto Oswaldo Cruz</journal-title></journal-title-group><issn pub-type="ppub">0074-0276</issn><issn pub-type="epub">1678-8060</issn><publisher><publisher-name>Instituto Oswaldo Cruz, Ministério da Saúde</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24714966</article-id><article-id pub-id-type="pmc">4015259</article-id><article-id pub-id-type="doi">10.1590/0074-0276130269</article-id><article-categories><subj-group subj-group-type="heading"><subject>Articles</subject></subj-group></article-categories><title-group><article-title>Silver nanoparticle production by the fungus
<italic>Fusarium oxysporum</italic>: nanoparticle characterisation and
analysis of antifungal activity against pathogenic yeasts</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Ishida</surname><given-names>Kelly</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="c01">+</xref></contrib><contrib contrib-type="author"><name><surname>Cipriano</surname><given-names>Talita Ferreira</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Rocha</surname><given-names>Gustavo Miranda</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Weissmüller</surname><given-names>Gilberto</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Gomes</surname><given-names>Fabio</given-names></name><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Miranda</surname><given-names>Kildare</given-names></name><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Rozental</surname><given-names>Sonia</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib></contrib-group><aff id="aff1"><label>1</label>Laboratório de Quimioterapia Antifúngica, Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP,<country>Brasil</country></aff><aff id="aff2"><label>2</label>Laboratório de Biologia Celular de Fungos</aff><aff id="aff3"><label>3</label>Laboratório de Física Biológica</aff><aff id="aff4"><label>4</label>Instituto Nacional de Metrologia, Padronização e Qualidade Industrial, Duque de Caxias, RJ,<country>Brasil</country></aff><aff id="aff5"><label>5</label>Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ,<country>Brasil</country></aff><author-notes><corresp id="c01"><label>+</label>Corresponding author: <email>ishidakelly@usp.br</email></corresp></author-notes><pub-date pub-type="epub"><day>19</day><month>11</month><year>2013</year></pub-date><pub-date pub-type="ppub"><month>4</month><year>2014</year></pub-date><volume>109</volume><issue>2</issue><fpage>220</fpage><lpage>228</lpage><history><date date-type="received"><day>17</day><month>5</month><year>2013</year></date><date date-type="accepted"><day>16</day><month>9</month><year>2013</year></date></history><permissions><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc/3.0/"><license-p>This is an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License, which permits
unrestricted non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.</license-p></license></permissions><abstract><p>The microbial synthesis of nanoparticles is a green chemistry approach that
combines nanotechnology and microbial biotechnology. The aim of this study was
to obtain silver nanoparticles (SNPs) using aqueous extract from the filamentous
fungus <italic>Fusarium oxysporum</italic> as an alternative to chemical
procedures and to evaluate its antifungal activity. SNPs production increased in
a concentration-dependent way up to 1 mM silver nitrate until 30 days of
reaction. Monodispersed and spherical SNPs were predominantly produced. After 60
days, it was possible to observe degenerated SNPs with in additional needle
morphology. The SNPs showed a high antifungal activity against
<italic>Candida</italic> and <italic>Cryptococcus</italic> , with minimum
inhibitory concentration values ≤ 1.68 µg/mL for both genera. Morphological
alterations of <italic>Cryptococcus neoformans</italic> treated with SNPs were
observed such as disruption of the cell wall and cytoplasmic membrane and lost
of the cytoplasm content. This work revealed that SNPs can be easily produced by
<italic>F. oxysporum</italic> aqueous extracts and may be a feasible,
low-cost, environmentally friendly method for generating stable and uniformly
sized SNPs. Finally, we have demonstrated that these SNPs are active against
pathogenic fungi, such as <italic>Candida</italic> and
<italic>Cryptococcus</italic> .</p></abstract><kwd-group><kwd>silver nanoparticle</kwd><kwd>microscopy</kwd><kwd>environmentally friendly procedure</kwd><kwd>antifungal</kwd><kwd><italic>Candida</italic></kwd><kwd><italic>Cryptococcus</italic></kwd></kwd-group><counts><fig-count count="5"></fig-count><table-count count="3"></table-count><ref-count count="32"></ref-count><page-count count="NaN"></page-count></counts></article-meta></front><body><p>Nanotechnology is a rapidly expanding field and have been potentially used in a wide
assortment of commercial products worldwide. Silver nanoparticles (SNPs) have attracted
specific attention due to their potential use in a range of applications, such as
electronics, biosensing, clothing manufacture, food storage, paints, sunscreens,
cosmetics and medical devices (<xref rid="B1" ref-type="bibr">Ahamed et al.
2010</xref>). SNPs have also a potent antibacterial and antifungal activity and general
anti-inflammatory effects. Furthermore, SNPs can improve wound healing and may be
exploited to develop dressings for wounds and burns and antibacterial coatings on
medical devices to reduce nosocomial infection rates (<xref rid="B1" ref-type="bibr">Ahamed et al. 2010</xref>, <xref rid="B5" ref-type="bibr">Chaloupka et al.
2010</xref>).</p><p>Although ultraviolet irradiation, aerosol technologies, lithography, laser ablation,
ultrasonic fields and photochemical reduction techniques have been used successfully to
produce nanoparticles, they remain expensive and involve the use of hazardous chemicals
(<xref rid="B25" ref-type="bibr">Narayanan & Sakthivel 2010</xref>). Therefore,
there is a growing effort to develop environmentally friendly methods for nanoparticle
production. The microbial synthesis of nanoparticles is a prime candidate approach
because it is a green chemistry method that combines nanotechnology and microbial
biotechnology (<xref rid="B25" ref-type="bibr">Narayanan & Sakthivel 2010</xref>).
Some microorganisms, including bacteria, yeast and filamentous fungi play an important
role in the remediation of toxic metals through the reduction of the metal ions;
therefore, these microorganisms could be employed as nanofactories for nanoparticle
production (<xref rid="B11" ref-type="bibr">Fortin & Beveridge 2000</xref>). Several
studies have shown that metal nanoparticles, such as gold, silver, gold-silver alloy,
selenium, tellurium, platinum, palladium, silica, titanium, zirconium, quantum dots and
magnetite can be biosynthesised by bacteria, actinomycetes, fungi and viruses [revised
by <xref rid="B25" ref-type="bibr">Narayanan and Sakthivel (2010)</xref>].</p><p>The fungus <italic>Fusarium oxysporum</italic> can reduce aqueous silver ions
extracellularly to generate SNPs (<xref rid="B2" ref-type="bibr">Ahmad et al.
2003</xref>). This process likely occurs through the action of both reductase
enzymes and electron shuttle quinones (<xref rid="B10" ref-type="bibr">Durán et al.
2005</xref>). In addition, biological nanoparticle synthesis often yields a more
consistent size distribution pattern than other methods due to direct stabilisation of
the nanoparticles by proteins involved in the synthesis process, as described by <xref rid="B10" ref-type="bibr">Durán et al. (2005)</xref>.</p><p><italic>Fusarium</italic> spp are filamentous fungi that are widely distributed in soil,
water, subterranean and aerial plant parts, plant debris and other organic substrates
(<xref rid="B26" ref-type="bibr">Nelson et al. 1994</xref>). The widespread
distribution of <italic>Fusarium</italic> spp is attributed to their ability to grow on
a wide range of substrates and their efficient dispersal mechanisms (<xref rid="B4" ref-type="bibr">Burgess 1981</xref>). Most species are harmless saprobes
and members of the soil microbial community; however, many species are mycotoxin
producers and are pathogenic to plants and humans.</p><p>In the present paper, we investigate the kinetics of SNPs production using an
environmentally friendly method of extracellular biosynthesis by <italic>F.
oxysporum</italic>. The characteristics and stability of the SNPs were evaluated by
electron and atomic force microscopy and the antifungal activity against pathogenic
species of <italic>Candida</italic> and <italic>Cryptococcus</italic> was evaluated by
broth microdilution and agar diffusion tests.</p><sec sec-type="materials|methods"><title>MATERIALS AND METHODS</title><p><italic>Fungal strains -</italic> The <italic>F. oxysporum</italic> 07 SD strain was
kindly provided by Elisa Espósito (Center for Environmental Sciences, University of
Mogi das Cruzes, Mogi das Cruzes, state of São Paulo, Brazil). The fungus was
cultivated and maintained on potato dextrose agar (PDA) (Becton & Dickinson and
Company, USA) at 28ºC in Petri dishes.</p><p>The following strains of <italic>Candida</italic> spp and
<italic>Cryptococcu</italic> s spp were used to evaluate the antifungal activity
of the SNPs: <italic>Candida albicans</italic> ATCC 10231, <italic>C.
albicans</italic> ATCC 24433, <italic>Candida krusei</italic> ATCC 6258,
<italic>Candida glabrata</italic> ATCC 2001, <italic>Candida
parapsilosis</italic> ATCC 22019, <italic>Candida tropicalis</italic> ATCC
13803, <italic>Cryptococcus neofor- mans</italic> ATCC 28957 and
<italic>Cryptococcus gattii</italic> ATCC 56990. The yeasts were maintained on
Sabouraud dextrose agar (SDA) (Becton & Dickinson) at 4ºC and subcultured at
least twice on the same medium at 35ºC for 48 h prior to use in experiments to
ensure optimal growth.</p><p><italic>SNP production - F. oxysporum</italic> previously grown on PDA at 28ºC was
inoculated in medium containing 2% malt extract and 0.5% yeast extract and incubated
at 28ºC for 7 days with agitation. Subsequently, the biomass was centrifuged, washed
three times with sterile water and weighed. Approximately 10 g of <italic>F.
oxysporum</italic> biomass was added to a glass Erlenmeyer flask containing 100
mL distilled water and incubated for 72 h at 28ºC with agitation. Then, the
components of the fungal aqueous extract were obtained by filtration through a 0.45
µm pore size nylon membrane filter and a silver nitrate (AgNO <sub>3</sub>) solution
was added to produce different concentrations of silver ions (0.5 mM, 1.0 mM, 1.5 mM
and 2.0 mM). The solutions were kept up to 60 days at room temperature in the dark.
Periodically, aliquots of the reaction solutions were removed and the absorption was
measured from 200-600 nm with a spectrophotometer (SpectraMax M2e, Molecular
Devices, Sunnyvale, CA, USA). The aliquots were also characterised using microscopy
methods as follows.</p><p><italic>SNP characterisation - Transmission electron microscopy (TEM)</italic> - A
drop of SNP colloidal solution obtained from a 1.0 mM AgNO <sub>3</sub> solution
collected at various times (5, 10, 15, 30 and 60 days) was directly dispensed on a
copper grid (300 mesh) coated with polyvinyl resin (FormVar, Ted Pella, Inc) and
dried in a vacuum desiccator. Images of the SNPs were obtained with a Jeol 1200
electron microscope (Jeol, Tokyo, Japan) equipped with a CCD Camera (MegaView III
model, Soft Image System, Germany) and processed with iTEM software (Soft Image
System). Routines were performed following the guidelines established by National
Institute of Standards and Technology (NIST) for size characterisation by
microscopy-based techniques (<xref rid="B15" ref-type="bibr">Jillavenkatesa et al.
2001</xref>). The SNPs size was measured using the software ImageJ [National
Institutes of Health (NIH), USA] (rsb.info.nih.gov/ij/) from several images obtained
by TEM. For elemental analysis, energy dispersive X-ray spectroscopy (EDX) was
performed. Briefly, X-rays were collected using a lithium-drifet silicon detector
with a Norvar window in a 0-10 keV energy range with a resolution of 10
eV/channel.</p><p><italic>Scanning electron microscopy (SEM)</italic> - A drop of SNP colloidal
solution obtained from a 1.0 mM AgNO <sub>3</sub> solution at various days of
incubation (5, 10, 15, 30 and 60 days) was directly adhered to coverslips coated
with FormVar and subsequently recovered by a carbon strip. The nanoparticles were
analysed by a backscattered electron detector with a QUANTA 250 scanning electron
microscope (FEI Company, Tokyo, Japan) operating at high vacuum mode.</p><p><italic>Atomic force microscopy</italic> - A 10 µL aliquot of SNP colloidal solution
obtained from a 1.0 mM AgNO <sub>3</sub> solution at various times (5, 10, 15, 30
and 60 days) was spread onto freshly cleaved 1 cm <sup>2</sup> mica slides and dried
with a low pressure jet of nitrogen gas. Next, the SNPs on the slide were examined
by an MFP-3D-BIO system (Asylum Research, Santa Barbara, CA, USA) operating in
intermittent contact mode using a cantilever Olympus AC240TS. SNP size analyses were
quantified and diameter size measured with the package particle analysis function in
ImageJ (NIH) using several SNPs images according to standards established by NIST (
<xref rid="B15" ref-type="bibr">Jillavenkatesa et al. 2001</xref> ).</p><p><italic>Antifungal activity assay -</italic> The in vitro antifungal activity of the
SNPs was evaluated using the disk difusion method (CLSI 2004) with some
modifications. This assay was used to test SNPs formed with different concentrations
of AgNO <sub>3</sub> solutions and at different time points after the start of the
reaction. Briefly, Müller-Hinton agar (Becton, Dickinson and Company) Petri plates
(4 mm of depth) were prepared and, subsequently, 100 µL of fungal inoculum [1-5 x 10
<sup>6</sup> colony-forming unit (CFU)/mL] was uniformly spread onto the plates.
Then, a 5 µL aliquot of SNP colloidal solution was impregnated on Whatman No. 1
sterile filter paper discs (5 mm in diameter). The discs were applied to the plates,
which were then incubated at 35ºC for 24-48 h. Finally, the inhibition halo was
measured. Water and aqueous fungal extract were used as negative controls for
antifungal activity.</p><p>In addition, the antifungal activity of the SNP colloidal solution obtained from a
30-day reaction with 1.0 mM AgNO <sub>3</sub> solution was evaluated by the broth
microdilution method (CLSI 2008). Briefly, the SNPs were serially diluted two-fold
in RPMI-1640 medium without sodium bicarbonate (Sigma Chemical Co, MO, USA) buffered
with 0.165 M MOPS (Sigma Chemical Co) in 96-well microtitre trays to obtain
concentrations of 0.1 to 54 µg/mL. A fungal suspension of 1-5 x 10 <sup>3</sup>CFU/mL was prepared and 100 µL was dispensed into each well to obtain final
concentrations of 0.5-2.5 x 10 <sup>3</sup> CFU/mL. The microtitre trays were
incubated at 35ºC for 48 h (<italic>Candida</italic> spp) or 72 h
(<italic>Cryptococcus</italic> spp) in a humidified chamber. The minimum
inhibitory concentration (MIC) was the lowest concentration of SNPs that resulted in
visual inhibition of fungal growth.</p><p>The determination of the minimum fungicidal concentration (MFC) was performed after
48 h of treatment with the inhibitory concentrations used in the broth microdilution
assay. An aliquot of all treatments were transferred onto SDA plates without the
presence of drugs. The plates were incubated at 35ºC for 48 h and the MFC was
determined. MFC means the lower concentration which showed no fungal growth.</p><p><italic>Morphological alterations - C. neoformans</italic> ATCC 28957 yeasts were
selected for the following experiments due to higher antifungal susceptibility
presenting the lowest SNPs MIC and MFC values. Yeasts were treated with a
sub-inhibitory concentration (0.21 µg/mL) of SNPs, for 72 h at 35ºC, were washed in
PBS, pH 7.2 and fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M
cacodylate buffer. Then, the yeasts were post-fixed in 1% osmium tetroxide in
cacodylate buffer containing 1.25% potassium ferrocyanide and 5 mM CaCl <sub>2</sub>for 2 h and serially dehydrated in ethanol. Subsequently, the yeasts were embedded
in Spurr’s epoxy resin and ultra-thin sections were obtained using a Reichert-Jung
Ultracut (Leica Microsystems, Wetzlar, Germany). Finally, the sections were stained
with 5% uranyl acetate and 0.5% lead citrate and visualised on a Jeol 1200 electron
microscope.</p><p><italic>Statistical analysis -</italic> Statistical analyses were performed using
Prism 5.0. One-way ANOVA (Dunnett’s test) was utilised to compare the SNP production
from different silver concentrations and Student’s <italic>t</italic> test was used
to analyse the SNP size. Samples were considered statistically significant when p
< 0.05.</p></sec><sec sec-type="results"><title>RESULTS</title><p><italic>SNP production -</italic> The production of SNPs was initially evaluated
visually for up to 60 days during the reaction of silver ions and aqueous extracts
from <italic>F. oxysporum</italic>. Darkening of the medium colour occurred during
the 60 days of the reaction for different silver ion concentrations compared to
aqueous extract from <italic>F. oxysporum</italic> without AgNO <sub>3</sub>solution and sterile distilled water. The darkening of the colloidal solution
occurred in a time-dependent manner for the first five days of the reaction; after
five days, the colour remained constant until 60 days (data not shown). These colour
changes are related to the reduction of silver ions that occurs during the
production of SNPs. At all this reaction times, the colour of the colloidal
solutions became darker in a dose-dependent manner until the concentration of 1.5 mM
AgNO <sub>3</sub>. SNP production was quantitatively measured by spectrophotometry.
SNPs exhibited the highest absorbance between 340-560 nm, with an absorption peak at
440 nm, for all AgNO <sub>3</sub> concentrations used and reaction times tested
(<xref ref-type="fig" rid="f01">Fig. 1</xref>). <xref ref-type="fig" rid="f02">Fig. 2</xref> shows that SNP production proceed the fastest rate from
zero-three days; production continued to increase up to 30 days and decrease between
30-60 days. Furthermore, SNP production increased dose-dependently way to the silver
ion concentration added to the reaction (<xref ref-type="fig" rid="f02">Fig.
2</xref>). Significantly fewer SNPs were produced from the 0.5 mM solution, as
evaluated by absorbance at 440 nm, than from ion silver concentrations above 1 mM (p
< 0.01, one-way ANOVA); this difference was most striking on the third day of the
reactions (p < 0.001, one-way ANOVA). Interestingly, there was no statistically
significant difference in SNP production at concentrations above 1 mM AgNO
<sub>3</sub>. For this reason, we used 1 mM AgNO <sub>3</sub> solutions for SNP
production in the following experiments characterising SNPs.</p><fig id="f01" orientation="portrait" position="float"><label>Fig. 1</label><caption><title>absorption spectrum from 20-600 nm of silver nanoparticles produced by
aqueous extracts of <italic>Fusarium oxysporum</italic> and several
concentrations of silver nitrate (AgNO <sub>3</sub>) solutions at different
times (0-60 days) after initiation of the reaction. A: 0.5 mM AgNO
<sub>3</sub>; B: 1.0 mM AgNO <sub>3</sub>; C: 1.5 mM AgNO <sub>3</sub> ;
D: 2.0 mM AgNO <sub>3</sub>.</title></caption><graphic xlink:href="0074-0276-mioc-109-02-00220-gf01"></graphic></fig><fig id="f02" orientation="portrait" position="float"><label>Fig. 2</label><caption><title>extracellular silver nanoparticles (SNPs) production by silver nitrate
solutions mixed with aqueous extract from <italic>Fusarium
oxysporum</italic> over time (up to 60 days). The absorption of the SNPs
was measured at 440 nm. *: p < 0.05; **: p < 0.01.</title></caption><graphic xlink:href="0074-0276-mioc-109-02-00220-gf02"></graphic></fig><p><italic>SNPs characterisation -</italic> Several microscopy techniques were used to
confirm SNP production, identify the presence of silver atoms and analyze SNP size
and morphology. TEM micrographs of five days SNPs revealed the presence of spherical
electron-dense nanoparticles that were frequently monodispersed and relatively
homogeneous in size, with variations in diameter from 3.4-26.8 nm (<xref ref-type="fig" rid="f03">Fig. 3A</xref>, <xref ref-type="table" rid="t01">Table
I</xref>). Maximum SNP diameter (64.9 nm) was observed on the 30th day (<xref ref-type="fig" rid="f03">Fig. 3B</xref>, <xref ref-type="table" rid="t01">Table
I</xref>).</p><fig id="f03" orientation="portrait" position="float"><label>Fig. 3</label><caption><title>transmission electron microscopy characterisation of silver nanoparticles
produced during different reaction periods. A: five days; B: 30 days; C: 60
days. Bars = 200 nm; D: the presence of silver atoms (black arrow) was
confirmed by energy-dispersive X-ray spectroscopy; Ag: silver; C: carbon;
Cu: copper; O: oxygen; P: phosphorus; S: sulphur; Si: silicon.</title></caption><graphic xlink:href="0074-0276-mioc-109-02-00220-gf03"></graphic></fig><table-wrap id="t01" orientation="portrait" position="float"><label>TABLE I</label><caption><title>Diameters of silver nanoparticles (SNPs) produced from 1.0 mM silver
nitrate in aqueous extracts of <italic>Fusarium oxysporum</italic> at
different reaction times</title></caption><table frame="hsides" rules="groups"><colgroup width="30%" span="1"><col width="40%" span="1"></col><col span="1"></col><col span="1"></col></colgroup><tbody><tr><td rowspan="3" colspan="1">Reaction time (days)</td><td align="center" colspan="2" rowspan="1">SNP diameter (nm)</td></tr><tr><td colspan="2" style="border-bottom: thin solid; border-color: #000000" rowspan="1"></td></tr><tr><td align="center" rowspan="1" colspan="1">Range</td><td align="center" rowspan="1" colspan="1">X ± SD</td></tr><tr><td colspan="3" style="border-bottom: thin solid; border-color: #000000" rowspan="1"></td></tr><tr><td rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">3.4-26.8</td><td align="center" rowspan="1" colspan="1">13.2 ± 4.2</td></tr><tr><td rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">4.8-56.6</td><td align="center" rowspan="1" colspan="1">21.5 ± 9.1</td></tr><tr><td rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">3.2-45.5</td><td align="center" rowspan="1" colspan="1">13.5 ± 7.6</td></tr><tr><td rowspan="1" colspan="1">30</td><td align="center" rowspan="1" colspan="1">4.8-64.9</td><td align="center" rowspan="1" colspan="1">27.3 ± 12.4</td></tr><tr><td rowspan="1" colspan="1">60</td><td align="center" rowspan="1" colspan="1">1.9-53.6</td><td align="center" rowspan="1" colspan="1">10.4 ± 10.1</td></tr></tbody></table><table-wrap-foot><p>SD: standard deviation; X: average.</p></table-wrap-foot></table-wrap><p>After 60 days of reaction, we observed needle-shaped nanoparticles in addition to the
spherical form previously observed. In addition, the spherical SNP diameter was very
heterogeneous varying from 1.9-53.6 nm (<xref ref-type="fig" rid="f03">Fig.
3C</xref>, <xref ref-type="table" rid="t01">Table I</xref>). There was observed
a significant difference in SNP size between all reactions times examined (p <
0.01), except between five-15 days. However, it is important to note that at five
days, the SNPs were more homogeneous in size than in 15 days and generally more
homogenous dispersed from each other. The presence of silver atoms in the colloidal
solution was confirmed by EDX (black arrow in <xref ref-type="fig" rid="f03">Fig.
3D</xref>).</p><p>Morphological analysis by AFM revealed two distinct structural arrangements of SNPs:
one of 300 ± 57 nm and another of 77 ± 30 nm (<xref ref-type="fig" rid="f04">Fig.
4B</xref>). The smaller structures observed by AFM include the spherical
electron-dense SNPs observed by TEM. The AFM images demonstrate that the particle
sizes are homogeneously distributed over the mica. In addition, medium-sized (50 nm)
SNPs were detected by SEM with a backscattered electron detector (<xref ref-type="fig" rid="f04">Fig. 4A</xref>). We also observed an amorphous material
surrounding some groups of nanoparticles.</p><fig id="f04" orientation="portrait" position="float"><label>Fig. 4</label><caption><title>microscopic characterisation of silver nanoparticles (SNPs) produced by
aqueous extracts of <italic>Fusarium oxysporum</italic> and 1.0 mM silver
nitrate over five days. A: the presence of metallic particles was detected
by scanning electron microscopy; B: the SNPS size and depth are indicated by
the colour scale at the side of the atomic force microscopy
micrograph.</title></caption><graphic xlink:href="0074-0276-mioc-109-02-00220-gf04"></graphic></fig><p><italic>Antifungal activity -</italic> All <italic>Candida</italic> species tested
were susceptible to the SNPs produced from AgNO <sub>3</sub> solutions with
concentrations above 1 mM. The results from the agar diffusion method revealed that
the inhibition halo diameter was similar for SNPs collected between five-60 days of
reaction (<xref ref-type="table" rid="t02">Table II</xref>). Among
<italic>Candida</italic> species, <italic>C. albicans</italic> ATCC 10231 was
the most susceptible to SNPs, as indicated by a growth inhibition halo of 13 mm when
incubated with SNPs produced from 1 mM AgNO <sub>3</sub> (~0.54 µg). By contrast,
<italic>C. krusei</italic> ATCC 2001 and <italic>C. tropicalis</italic> ATCC
13803 were less susceptible to SNPs. Similar patterns of growth inhibition were
observed for the <italic>Cryptococcus</italic> species (<xref ref-type="table" rid="t02">Table II</xref>).</p><table-wrap id="t02" orientation="portrait" position="float"><label>TABLE II</label><caption><title>Growth inhibition halos (mm) of <italic>Candida</italic> spp and
<italic>Cryptococcus</italic> spp after 48 h of incubation in contact
with a paper disc impregnated with silver nanoparticle colloidal solutions
produced from different concentrations of silver nitrate (AgNO <sub>3</sub>)
with different reaction times</title></caption><table frame="hsides" rules="groups"><colgroup width="10%" span="1"><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col></colgroup><tbody><tr><td rowspan="1" colspan="1">Reaction time (days)</td><td align="center" rowspan="1" colspan="1">AgNO <sub>3</sub>concentration (mM)</td><td align="center" rowspan="1" colspan="1"><italic>Candida</italic><italic>albicans</italic> ATCC 10231</td><td align="center" rowspan="1" colspan="1"><italic>C. albicans</italic> ATCC 24433</td><td align="center" rowspan="1" colspan="1"><italic>Candida</italic><italic>parapsilosis</italic> ATCC22019</td><td align="center" rowspan="1" colspan="1"><italic>Candida</italic><italic>tropicalis</italic> ATCC 13803</td><td align="center" rowspan="1" colspan="1"><italic>Candida krusei</italic> ATCC 6258</td><td align="center" rowspan="1" colspan="1"><italic>Candida glabrata</italic> ATCC 2001</td><td align="center" rowspan="1" colspan="1"><italic>Cryptococcus neoformans</italic> ATCC 28957</td><td align="center" rowspan="1" colspan="1"><italic>Cryptococcus gattii</italic> ATCC 56990</td></tr><tr><td colspan="10" style="border-bottom: thin solid; border-color: #000000" rowspan="1"></td></tr><tr><td rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">9</td></tr><tr><td rowspan="1" colspan="1">30</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td rowspan="1" colspan="1">60</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">8</td></tr></tbody></table></table-wrap><p>The broth microdilution method revealed that the <italic>Cryptococcus</italic>species were slightly more susceptible to SNP treatment (MICs from 0.42-0.84 µg/mL)
than the <italic>Candida</italic> species (MICs from 0.84-1.68 µg/mL) (<xref ref-type="table" rid="t03">Table III</xref>). In addition, SNPs exhibited
fungicidal activity (with MFC values up to 4 times the MIC value) against most of
the species tested, with the exception of <italic>C. parapsilosis</italic> ATCC
22019 and <italic>C. krusei</italic> ATCC 6258 (<xref ref-type="table" rid="t03">Table III</xref>). These results are in agreement with those obtained using the
agar diffusion method.</p><table-wrap id="t03" orientation="portrait" position="float"><label>TABLE III</label><caption><title>Minimum inhibitory concentration (MIC) and minimum fungicidal
concentration (MFC) of silver nanoparticles (SNPs) for
<italic>Candida</italic> spp and <italic>Cryptococcus</italic> spp
concentrations</title></caption><table frame="hsides" rules="groups"><colgroup width="25%" span="1"><col span="1"></col><col span="1"></col><col span="1"></col><col span="1"></col></colgroup><tbody><tr><td rowspan="1" colspan="1">Strains</td><td align="center" rowspan="1" colspan="1">MIC (µg/mL)</td><td align="center" rowspan="1" colspan="1">MFC (µg/mL)</td><td align="center" rowspan="1" colspan="1">MFC/MIC</td></tr><tr><td colspan="4" style="border-bottom: thin solid; border-color: #000000" rowspan="1"></td></tr><tr><td rowspan="1" colspan="1"><italic>Cryptococcus neoformans</italic> ATCC 28957</td><td align="center" rowspan="1" colspan="1">0.42</td><td align="center" rowspan="1" colspan="1">0.42</td><td align="center" rowspan="1" colspan="1">1</td></tr><tr><td rowspan="1" colspan="1"><italic>Cryptococcus gattii</italic> ATCC 56990</td><td align="center" rowspan="1" colspan="1">0.84</td><td align="center" rowspan="1" colspan="1">0.84</td><td align="center" rowspan="1" colspan="1">1</td></tr><tr><td rowspan="1" colspan="1"><italic>Candida glabrata</italic> ATCC 2001</td><td align="center" rowspan="1" colspan="1">1.68</td><td align="center" rowspan="1" colspan="1">1.68</td><td align="center" rowspan="1" colspan="1">1</td></tr><tr><td rowspan="1" colspan="1"><italic>Candida albicans</italic> ATCC 10231</td><td align="center" rowspan="1" colspan="1">1.68</td><td align="center" rowspan="1" colspan="1">3.4</td><td align="center" rowspan="1" colspan="1">2</td></tr><tr><td rowspan="1" colspan="1"><italic>C. albicans</italic> ATCC 24433</td><td align="center" rowspan="1" colspan="1">1.68</td><td align="center" rowspan="1" colspan="1">3.4</td><td align="center" rowspan="1" colspan="1">2</td></tr><tr><td rowspan="1" colspan="1"><italic>Candida tropicalis</italic> ATCC 13803</td><td align="center" rowspan="1" colspan="1">1.68</td><td align="center" rowspan="1" colspan="1">3.4</td><td align="center" rowspan="1" colspan="1">2</td></tr><tr><td rowspan="1" colspan="1"><italic>Candida krusei</italic> ATCC 6258</td><td align="center" rowspan="1" colspan="1">0.84</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">15</td></tr><tr><td rowspan="1" colspan="1"><italic>Candida parapsilosis</italic> ATCC 22019</td><td align="center" rowspan="1" colspan="1">0.84</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">15</td></tr></tbody></table></table-wrap><p><italic>Morphological alterations of C. neoformans treated with SNPs</italic> -
Untreated <italic>C. neoformans</italic> ATCC 28957 yeasts exhibited a
well-preserved cellular ultrastructure with a compact cell wall (CW), continuous
cytoplasmic membrane, homogeneous and electron-dense cytoplasm and polysaccharide
capsule (<xref ref-type="fig" rid="f05">Fig. 5A</xref>, <xref ref-type="fig" rid="f05">B</xref>). By contrast, yeasts treated with sub-inhibitory
concentrations of SNPs (0.21 μg/mL), for 72 h at 35ºC, exhibited a disrupted CW and
several invaginations in the cytoplasmic membrane (<xref ref-type="fig" rid="f05">Fig. 5C</xref>, <xref ref-type="fig" rid="f05">D</xref>). In addition,
increased CW thickness was observed (<xref ref-type="fig" rid="f05">Fig. 5E</xref>,
<xref ref-type="fig" rid="f05">F</xref>). Interestingly, some SNPs appear to be
retained in the fungal capsule (black arrowhead in <xref ref-type="fig" rid="f05">Fig. 5F</xref>).</p><fig id="f05" orientation="portrait" position="float"><label>Fig. 5</label><caption><title>morphological alterations of <italic>Cryptococcus neoformans</italic>treated with sub-inhibitory concentrations of silver nanoparticles (SNPs)
(0.21 µg/mL) for 72 h at 35ºC. The untreated yeast exhibit a compact cell
wall (CW), continuous cytoplasmic membrane (cm), homogeneous and
electron-dense cytoplasm and a polysaccharide capsule (c) surrounding the
cell (A, B). By contrast, yeasts treated with SNPs had a disrupted
cytoplasmic membrane and CW (asterisk) and increased cell wall thickness
(CW) (C, D). The SNPs appear to be retained in the polysaccharide capsule
(F, black arrowhead).</title></caption><graphic xlink:href="0074-0276-mioc-109-02-00220-gf05"></graphic></fig></sec><sec sec-type="discussion"><title>DISCUSSION</title><p>In the last decade, the development of biological systems as an environmentally
friendly method for metal nanoparticle formation has emerged as an interesting and
important scientific field. A wide number of microorganisms, including bacteria,
yeast, filamentous fungi, algae and plants, have been shown to be capable of
fabricating various types of metal nanoparticles like silver, gold, palladium and
others (<xref rid="B28" ref-type="bibr">Quester et al. 2013</xref>).</p><p>Previous studies have demonstrated that filamentous fungi, such as <italic>F.
oxysporum</italic> (<xref rid="B10" ref-type="bibr">Durán et al. 2005</xref>),
<italic>Fusarium accuminatum</italic> (<xref rid="B14" ref-type="bibr">Ingle et
al. 2008</xref>), <italic>Aspergillus niger</italic> (<xref rid="B12" ref-type="bibr">Gade et al. 2008</xref>), <italic>Amylomyces rouxii</italic> (<xref rid="B24" ref-type="bibr">Musarrat et al. 2010</xref>) and the endophytic fungus
<italic>Epicoccum nigrum</italic> (<xref rid="B27" ref-type="bibr">Qian et al.
2013</xref>) are most efficient at producing SNPs. However, the majority of
these studies used only a single concentration of AgNO <sub>3</sub> solution (1.0
mM) to produce SNPs. Our work further characterises the production of SNPs by a
green synthesis method using aqueous extracts of <italic>F. oxysporum</italic> and
different silver concentrations for up to 60 days. Spectrophotometric analyses
revealed that concentrations below 1 mM AgNO <sub>3</sub> produced fewer SNPs than
concentrations ≥ 1 mM AgNO <sub>3</sub> . Interestingly, the use of concentrations
> 1.0 mM did not result in increased SNP production. With respect to the kinetics
of SNP production, we found that increasing amounts of SNPs were produced for the
first 30 days, followed by a decrease in production after 60 days. This finding
indicates that the SNPs likely began to degrade after a prolonged incubation time.
These changes in SNP production rates correlated with the appearance of
needle-shaped SNPs, in addition to the previously observed spherical forms, on day
60 of the reactions. SNPs sizes could range from 1-100 nm and are characterised by a
large surface area to volume ratio (<xref rid="B29" ref-type="bibr">Rai et al.
2009</xref>). The SNPs produced in this work were predominantly monodispersed
and spherical, with diameters ranging from 1.9-64.9 nm. Our data revealed that the
optimal SNP production occurred when a 1.0 mM AgNO <sub>3</sub> solution was used
during three-five days into the reaction. In this case homogenous SNPs with
approximate diameters of 10 nm were detected.</p><p>The reducing agent, reaction medium and SNP stabilisation are three key factors in
the synthesis of metallic nanoparticles (<xref rid="B20" ref-type="bibr">Liu et al.
2009</xref>). According to <xref rid="B10" ref-type="bibr">Durán et al.
(2005)</xref>, reductases in the aqueous extracts of <italic>F.
oxysporum</italic> are responsible for the reduction of Ag cations and
subsequent SNP production. In addition, the SNP size, spherical form stability and
dispersion may be mediated by an interaction between the SNPs and proteins present
in the fungal extract (<xref rid="B10" ref-type="bibr">Durán et al. 2005</xref>).
Furthermore, SNPs formed in solutions of complex organic molecules seem to be
stabilised by a protective coating. <xref rid="B3" ref-type="bibr">Akaighe et al.
(2011)</xref> reported that humic acid facilitate the generation of SNPs by
reducing silver ions, which suggests that nanoparticles can exist in a natural
environment. Humic acid appears to stabilise the nanoparticles by coating them and
preventing their aggregation into a larger mass of silver (<xref rid="B3" ref-type="bibr">Akaighe et al. 2011</xref>). The fungal extract used in this work could
also be considered a natural environment. This could explain the difference in the
diameters of the agglomerates observed by AFM and TEM. TEM cannot resolve the large
agglomerates of SNPs as well as AFM (300 ± 57 nm). Furthermore, the TEM and SEM
backscattered images only reveal the silver atomic nuclei.</p><p>Previous studies have shown that SNPs exhibit antimicrobial activity against
different bacterial species, such as <italic>Shigella dysenteriae</italic> type I,
<italic>Staphylococcus aureus</italic>, <italic>Citrobacter</italic> sp.,
<italic>Escherichia coli</italic>, <italic>Pseudomonas aeruginosa</italic> and
<italic>Bacillus subtilis</italic> (<xref rid="B24" ref-type="bibr">Musarrat et
al. 2010</xref>) and some fungal species, such as <italic>Trichophyton
mentagrophytes</italic> and <italic>Candida</italic> spp (<xref rid="B17" ref-type="bibr">Keuk-Jun et al. 2008</xref>, <xref rid="B24" ref-type="bibr">Musarrat
et al. 2010</xref>). In our work, we employed two different methods to determine
the susceptibility of <italic>Candida</italic> spp and <italic>Cryptococcus</italic>spp. The disc diffusion method resulted in the formation of zones of growth
inhibition ranging from 8-15 mm and with the broth microdilution assay we observed
MICs of 0.42-1.68 µg/mL.</p><p>Similar results were previously reported by <xref rid="B24" ref-type="bibr">Musarrat
et al. (2010)</xref> and <xref rid="B16" ref-type="bibr">Kaviya et al.
(2011)</xref>, who tested the effect of SNPs on bacteria and fungi using the
same methods. <xref rid="B17" ref-type="bibr">Keuk-Jun et al. (2008)</xref> observed
that the MICs of SNPs against <italic>C. albicans</italic> were lower (2-4 µg/mL)
than against <italic>Candida</italic> non- <italic>albicans</italic> (1-25 µg/mL).
Recently, <xref rid="B27" ref-type="bibr">Qian et al. (2013)</xref> and <xref rid="B31" ref-type="bibr">Xu et al. (2013)</xref> observed SNPs antifungal
activity against several fungi as <italic>Candida</italic> spp,
<italic>Aspergillus</italic> spp, <italic>Fusarium</italic> spp <italic>, C.
neoformans</italic> and <italic>Sporothrix schenckii</italic> presenting MIC
values of 0.12-1 µg/mL.</p><p>In our work, we observed similar MIC values for all <italic>Candida</italic> spp and
<italic>Cryptococcus</italic> spp tested. However, the MFC values suggest that
SNPs are fungistatic to <italic>C. parapsilosis</italic> and <italic>C.
krusei</italic> and fungicidal to <italic>C. albicans</italic>, <italic>C.
tropicalis</italic>, <italic>C. glabrata</italic> and
<italic>Cryptococcus</italic> spp. Recently, <xref rid="B22" ref-type="bibr">Monteiro et al. (2011)</xref> reported an inhibitory effect of SNPs on
<italic>C. albicans</italic> and <italic>C. glabrata</italic> biofilm formation
due to reduced cell viability.</p><p>Our ultrastructural studies revealed that the yeast cell alterations induced by SNPs
treatment largely occur in the cytoplasmic membrane and fungal CW. <italic>C.
neoformans</italic> exposed to SNPs exhibit cytoplasmic leakage and apparent SNP
retention in the polysaccharide capsule. To our knowledge, this is the first report
that demonstrates the action of SNPs on <italic>C. neoformans</italic> cell
envelope. Previous work has already demonstrated the ultrastructural effect on
<italic>C. albicans</italic> yeast treated with SNPs (<xref rid="B18" ref-type="bibr">Kim et al. 2009</xref>). SNPs also presented inhibitory activity
against <italic>C. albicans</italic> (MIC = 2 µg/mL) and the action may exert the
disrupting of the structure of the <italic>C. albicans</italic> CW and cytoplasm
membrane and inhibit the normal budding process due to the destruction of the
membrane integrity when yeasts were treated with SNPs concentration above MIC value
(170 µg/mL) (<xref rid="B18" ref-type="bibr">Kim et al. 2009</xref>). Our work
treated <italic>C. albicans</italic> yeasts with MIC/2 concentration of SNPs,
however none ultrastructural alterations was observed by TEM technique.
Interestingly, the MIC/2 value of SNPs was able to lead a change of cell envelop
ultrastructure of <italic>C. neoformans</italic>.</p><p>Several other studies have demonstrated that the cell envelope constitutes the major
target of SNP antimicrobial activity. <xref rid="B7" ref-type="bibr">Chwalibog et
al. (2010)</xref> also demonstrated that the SNPs can self-assemble and interact
with <italic>C. albicans</italic> and <italic>S. aureus</italic> cells, leading to
the disintegration of CWs and cytoplasmic membranes and cytoplasm leakage. The
effect of SNPs on <italic>S. aureus</italic> growth, morphology and CW integrity was
investigated (<xref rid="B21" ref-type="bibr">Mirzajani et al. 2011</xref>),
revealing bacterial growth inhibition, CW damage with peptidoglycan variations and
release of muramic acid and accumulation of SNPs in the bacterial membrane at SNP
concentrations greater than 8 µg/mL. Membrane disruption by SNPs is likely due to
the production of reactive oxygen species (ROS), including free radicals that cause
membrane lipid peroxidation. This membrane disruption allows the passage of SNPs
into the cytoplasm, which causes subsequent damage of DNA and other
phosphorus-containing compounds and impairs the respiratory chain and cell division
(<xref rid="B29" ref-type="bibr">Rai et al. 2009</xref>). Recently, <xref rid="B13" ref-type="bibr">Hwang et al. (2012)</xref> showed SNPs possess
antifungal effects through apoptosis. The treatment induced in <italic>C.
albicans</italic> yeasts an accumulation of ROS, reduction in the mitochondrial
membrane potential, phosphatidylserine externalisation, DNA and nuclear
fragmentation and the activation of metacaspases.</p><p>In mammalian cells, cytotoxicity is related to a loss of cell viability, DNA
fragmentation and subsequent apoptosis. Furthermore, the endoplasmic reticulum may
play an important role in the response to oxidative stress-induced damage and is
quite sensitive to oxidative damage (<xref rid="B32" ref-type="bibr">Zhang et al.
2012</xref>). Interestingly, biogenic SNPs seems to be generally less
cyto/genotoxic in vivo compared with chemically synthesised nanoparticles.
Furthermore, human cells were found to have a greater resistance to the toxic
effects of SNPs in comparison with other organisms (<xref rid="B19" ref-type="bibr">Lima et al. 2012</xref>).</p><p>The antimicrobial effects of SNPs depend on their size and the rate of silver ion
release. Smaller-sized particles exhibit greater activity due to their higher
surface area to volume ratio (<xref rid="B23" ref-type="bibr">Morones et al.
2005</xref>). The ionic form of silver has been known for centuries to cure
infectious diseases caused by various bacterial species, e.g., <italic>E.
coli</italic>, <italic>S. aureus</italic>, <italic>Klebsiella</italic> sp. and
<italic>Pseudomonas</italic> sp. (<xref rid="B6" ref-type="bibr">Chopra
2007</xref>, <xref rid="B29" ref-type="bibr">Rai et al. 2009</xref>). SNPs have
an advantage over ionic silver because they exhibit reduced toxicity and greater
antimicrobial potential (<xref rid="B14" ref-type="bibr">Ingle et al. 2008</xref>).
The superior antimicrobial properties of SNPs compared to silver salts are due to
the large surface area of the particles, which provides better contact with the
microorganisms. In addition, the SNPs release silver ions directly into the
bacteria, which enhance bactericidal activity (<xref rid="B29" ref-type="bibr">Rai
et al. 2009</xref>).</p><p>Although in vitro and in vivo studies have indicated that SNPs are toxic to mammalian
cells and that increased exposure of humans to SNPs entails potential risk, some
studies have described broad applications of SNPs in the technological and medical
fields (<xref rid="B2" ref-type="bibr">Ahmad et al. 2003</xref>, <xref rid="B5" ref-type="bibr">Chaloupka et al. 2010</xref>, <xref rid="B30" ref-type="bibr">Seil & Webster 2012</xref>). However these applications remain
largely unexplored. The production of SNPs using aqueous extracts of the fungus
<italic>F. oxysporum</italic> is a potential candidate for low-cost and
environmentally friendly production of stable and uniformly sized SNPs with
anticandidal and anticryptococcal activities. In this regard, the results obtained
in this work open several avenues of further study, such as purification and
biochemical characterisation of reductases produced by <italic>F. oxysporum</italic>or development of an alternative SNP formulation that reduces the toxicity of
silver. In addition, the utilisation of silver metal in nanoparticulate form may be
a new strategy for the treatment of fungal infection.</p></sec></body><back><fn-group><fn fn-type="supported-by"><p>Financial support: CNPq, CAPES, FAPERJ</p></fn></fn-group><ref-list><title>REFERENCES</title><ref id="B1"><element-citation publication-type="journal"><person-group><name><surname>Ahamed</surname><given-names>M</given-names></name><name><surname>AlSalhi</surname><given-names>MS</given-names></name><name><surname>Siddiqui</surname><given-names>MKJ</given-names></name></person-group><year>2010</year><article-title>Silver nanoparticle applications and human
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