research article growth kinetics and mechanistic action of...

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Research Article Growth Kinetics and Mechanistic Action of Reactive Oxygen Species Released by Silver Nanoparticles from Aspergillus niger on Escherichia coli Shivaraj Ninganagouda, Vandana Rathod, Dattu Singh, Jyoti Hiremath, Ashish Kumar Singh, Jasmine Mathew, and Manzoor ul-Haq Department of Microbiology, Gulbarga University, Gulbarga, Karnataka 585106, India Correspondence should be addressed to Vandana Rathod; drvandanarathod@rediffmail.com Received 21 February 2014; Revised 13 May 2014; Accepted 18 May 2014; Published 16 June 2014 Academic Editor: Ming-Fa Hsieh Copyright © 2014 Shivaraj Ninganagouda et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Silver Nanoparticles (AgNPs), the real silver bullet, are known to have good antibacterial properties against pathogenic microorganisms. In the present study AgNPs were prepared from extracellular filtrate of Aspergillus niger. Characterization of AgNPs by UV-Vis spectrum reveals specific surface plasmon resonance at peak 416 nm; TEM photographs revealed the size of the AgNPs to be 20–55nm. Average diameter of the produced AgNPs was found to be 73nm with a zeta potential that was 24 mV using Malvern Zetasizer. SEM micrographs showed AgNPs to be spherical with smooth morphology. EDS revealed the presence of pure metallic AgNPs along with carbon and oxygen signatures. Of the different concentrations (0, 2.5, 5, 10, and 15 g/mL) used 10 g/mL were sufficient to inhibit 10 7 CFU/mL of E. coli. ROS production was measured using DCFH-DA method and the the free radical generation effect of AgNPs on bacterial growth inhibition was investigated by ESR spectroscopy. is paper not only deals with the damage inflicted on microorganisms by AgNPs but also induces cell death through the production of ROS released by AgNPs and also growth kinetics of E. coli supplemented with AgNPs produced by A. niger. 1. Introduction e broad spectrum antimicrobial properties of silver nanoparticles (AgNPs) encourage its use in biomedical appli- cations; with the rapid development of nanobiotechnology, applications have been extended further and now silver is the engineered nanomaterial most commonly used in consumer products [1, 2]. It has been known that silver and its compounds have strong inhibitory and bactericidal effects as well as broad spectrum of antimicrobial activities for bacteria [35], fungi [6], and viruses [7]. Compared with other metals, silver exhibits higher toxicity to microorganisms while it exhibits lower toxicity to mammalian cells [8]. AgNPs had been known for a long time but have been paid little attention. Advances in recent research on nanobiotechnology appear to revive the potential of AgNPs for antimicrobial applications [9]. Nanoparticles are used in many applications as they exhibit interesting and useful properties, which may be very different to their parent bulk material, because nanoparticles have extremely high surfaced area to volume ratios, so the properties of the nanoparticles are dominated by surface atom contribution [10]. Biologically inspired nanoparticle syntheses are currently a rapid expanding area of research which draws on many different disciplines. Recently, there has been lots of interests in biologically controlled synthesis as a greener, cheaper alternative to other methods [11]. e use of fungi in the synthesis of AgNPs is a relatively recent addition and holds promise for large scale nanopar- ticles production. In fact, fungi secrete large amount of the enzymes involved in AgNPs synthesis and are simpler to grow both in laboratory and at industrial scale [12]. In the present study we emphasis on biosynthesis of AgNPs from extracellular filtrate of Aspergillus niger and study the mechanistic action of reactive oxygen species (ROS) generated by AgNPs through electron spin resonance spectroscopy (ESR) from the surface of Ag + on pathogenic Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 753419, 9 pages http://dx.doi.org/10.1155/2014/753419

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Page 1: Research Article Growth Kinetics and Mechanistic Action of ...downloads.hindawi.com/journals/bmri/2014/753419.pdf · Species Released by Silver Nanoparticles from Aspergillus niger

Research ArticleGrowth Kinetics and Mechanistic Action of Reactive OxygenSpecies Released by Silver Nanoparticles from Aspergillus nigeron Escherichia coli

Shivaraj Ninganagouda Vandana Rathod Dattu Singh Jyoti HiremathAshish Kumar Singh Jasmine Mathew and Manzoor ul-Haq

Department of Microbiology Gulbarga University Gulbarga Karnataka 585106 India

Correspondence should be addressed to Vandana Rathod drvandanarathodrediffmailcom

Received 21 February 2014 Revised 13 May 2014 Accepted 18 May 2014 Published 16 June 2014

Academic Editor Ming-Fa Hsieh

Copyright copy 2014 Shivaraj Ninganagouda et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Silver Nanoparticles (AgNPs) the real silver bullet are known to have good antibacterial properties against pathogenicmicroorganisms In the present study AgNPs were prepared from extracellular filtrate of Aspergillus niger Characterization ofAgNPs by UV-Vis spectrum reveals specific surface plasmon resonance at peak 416 nm TEM photographs revealed the size of theAgNPs to be 20ndash55 nm Average diameter of the produced AgNPs was found to be 73 nm with a zeta potential that was minus24mVusing Malvern Zetasizer SEM micrographs showed AgNPs to be spherical with smooth morphology EDS revealed the presenceof pure metallic AgNPs along with carbon and oxygen signatures Of the different concentrations (0 25 5 10 and 15120583gmL) used10 120583gmL were sufficient to inhibit 107 CFUmL of E coli ROS production was measured using DCFH-DA method and the thefree radical generation effect of AgNPs on bacterial growth inhibition was investigated by ESR spectroscopy This paper not onlydeals with the damage inflicted on microorganisms by AgNPs but also induces cell death through the production of ROS releasedby AgNPs and also growth kinetics of E coli supplemented with AgNPs produced by A niger

1 Introduction

The broad spectrum antimicrobial properties of silvernanoparticles (AgNPs) encourage its use in biomedical appli-cations with the rapid development of nanobiotechnologyapplications have been extended further and now silveris the engineered nanomaterial most commonly used inconsumer products [1 2] It has been known that silver and itscompounds have strong inhibitory and bactericidal effects aswell as broad spectrum of antimicrobial activities for bacteria[3ndash5] fungi [6] and viruses [7] Comparedwith othermetalssilver exhibits higher toxicity to microorganisms while itexhibits lower toxicity to mammalian cells [8] AgNPs hadbeen known for a long time but have been paid little attentionAdvances in recent research on nanobiotechnology appear torevive the potential of AgNPs for antimicrobial applications[9]

Nanoparticles are used in many applications as theyexhibit interesting and useful properties which may be very

different to their parent bulk material because nanoparticleshave extremely high surfaced area to volume ratios so theproperties of the nanoparticles are dominated by surfaceatom contribution [10] Biologically inspired nanoparticlesyntheses are currently a rapid expanding area of researchwhich draws on many different disciplines Recently therehas been lots of interests in biologically controlled synthesisas a greener cheaper alternative to other methods [11]

The use of fungi in the synthesis of AgNPs is a relativelyrecent addition and holds promise for large scale nanopar-ticles production In fact fungi secrete large amount of theenzymes involved inAgNPs synthesis and are simpler to growboth in laboratory and at industrial scale [12]

In the present study we emphasis on biosynthesis ofAgNPs from extracellular filtrate of Aspergillus niger andstudy the mechanistic action of reactive oxygen species(ROS) generated by AgNPs through electron spin resonancespectroscopy (ESR) from the surface of Ag+ on pathogenic

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 753419 9 pageshttpdxdoiorg1011552014753419

2 BioMed Research International

Escherichia coli and confirm the obtained transmission elec-tron microscopy (TEM) results by Kinetic studies throughgrowth curve

2 Materials and Methods

21 Biosynthesis and Characterization of AgNPs The fungusA niger was grown in 250mL Erlenmeyer flasks containingmalt extract glucose yeast extract and peptone (MGYP-03 1 03 and 05 and pH 6) at temperature 29∘CAfter incubation mycelium was separated by filtrationwashed with sterile distilled water to remove traces of mediacontents and resuspended in 100mL distilled water for about48 hrs The suspension was filtered through Whatman filterpaper number 1 The cell filtrate was challenged with silvernitrate (1mM) for AgNPs biosynthesis

The Optical density of the synthesized AgNPs was char-acterized by UV-Vis spectroscopy (T 90+ UV-VIS spec-trophotometer) the interaction between protein and AgNPswas evaluated using Fourier-transform infrared spectroscopy(FTIR) (Perkin Elmer model 783 spectrophotometer) thesize and morphology of the AgNPs were examined bytransmission electron microscopy (TEM) (Hitachi H 7500ID Japan) the particle size distribution of AgNPs wasevaluated using Malvern Zetasizer nanoseries spectrometer(Malvern Instruments Ltd) shape and surface morphologyof nanoparticleswas characterized by using scanning electronmicroscopy (SEM) (JEOLModel JSM-6390 LV) the presenceof elemental silver was confirmed through energy dispersivespectroscopy (EDS) (JEOL Model JED-2300) and release ofreactive oxygen species (ROS) from the surface of AgNPs wasevaluated by employing electron spin resonance spectroscopy(ESR) (JES-FA 200ESR Spectrometer JEOL Japan)

22 Bactericidal Activity of AgNPs Agar dilution methodwas used to study the bactericidal activity of AgNPs onE coli Nutrient Agar (NA) supplemented with variousconcentrations ofAgNPs (0 25 5 10 and 15120583gmL) and eachplate was inoculated with 107 CFUmL of E coli by spreadplating Plates inoculatedwith E coli but without AgNPswereused as control Number of surviving bacteria in agar plateswas counted after 24 hours of incubation at 37∘C [13 14]

23 Bacterial GrowthKinetics against Different Concentrationsof AgNPs To study the bacterial growth curve E coli culturewas inoculated with fresh colonies and incubated for 12 hourovernight at 37∘C in nutrient broth (NB) Bacterial growthcurves were determined by measuring the optical density(OD) at 600 nm using a spectrophotometer At this thejuncture theODobtainedwas 10 To different concentrations(0ndash15120583gmL) ofAgNPs the aforesaidE coli culturewas addedand the turbidity was measured at different time intervals (05 10 15 20 and 25 hrs) [14] Experiment was repeated thrice

24 Release of ROS from the Surface of AgNPs and Its Mech-anism The interaction of AgNPs with E coli was assessedusing TEM Broth containing E coli exposed to AgNPswas subjected to TEM at a time intervals of 1 5 8 and

12 hrs respectively The samples which were subjected toTEM studies were also sent for ESR studies to detect thefree radical generation from the surface of silver ESR is ananalytical method to detect the free radical generated fromthe surface of silver It is based on absorption of microwaveradiation by an unpaired electron when it is exposed toa strong magnetic field AgNPs that contain free radicalstherefore are detected by ESR Free radical generation fromthe surface of Agwas recorded using ESR spectrophotometerThe generation of ROS from AgNPs was measured using2101584071015840-dichlorofluorescein diacetate (DCFH-DA) [14] 2101584071015840-Dichlorodihydrofluorescein diacetate (DCFH-DA) is one ofthe most widely used techniques for directly measuring theredox state of a cell DCFH-DA a cell permeable nonfluores-cent precursor of DCF can be used as an intracellular probefor oxidative stress It has many advantages over other tech-niques developed as it is very easy to use extremely sensitiveto changes in the redox state of a cell inexpensive and canbe used to follow changes in ROS over time 107 CFUmL ofcells were treated with 20120583gmL of AgNPs and incubated at37∘C for 5 h then centrifuged at 4∘C for 15min at 600timesg andthe obtained supernatantwas treatedwith 100120583MDCFH-DAfor 1 h The ROS formed was measured using Fluorescencespectrometry

To check the inhibition of E coli targeted through theROS produced by AgNPs from A niger we conducted a sep-arate experiment using ascorbic acid as an antioxidant whichacts as a scavenger NA plates supplemented with AgNPs(10 120583gmL) were also incorporated with 10mM ascorbic acidas a scavenger Thus the plates were inoculated with fresh12 hrs cultures of E coli and the surviving rate was countedafter 24 hrs of incubation [15ndash17]

3 Results and Discussion

31 Biosynthesis and Characterization of AgNPs The produc-tion of AgNPs by A niger was indicated by the appearance ofbrown color in the reaction mixture The UV-Vis spectrumfor AgNPs is obtained by exposing the sample to UV-lightfrom a light sourceThe specific surface plasmon resonance isresponsible for their unique remarkable optical phenomenonA single peak with a maximum of 416 nm corresponding tothe surface plasmon resonance of AgNPs was observed inthe UV-Vis spectrum (Figure 1) Singh et al [3] reported theextracellular biosynthesis of AgNPs using endophytic fungusPenicillium sp at the maximum absorbance peak of 425 nmand also reported that increase in concentration of silvernitrate increases the particle size due to aggregation of largerAgNPs In an another study Sondi and Salopek-Sondi [13]reported the surface Plasmon band at 405 nm at this peak theappearance of brown color clearly indicates the formation ofAgNPs

The interaction between protein andAgNPswas analyzedby Fourier-transform infrared spectroscopy (FTIR) TheAgNPs suspensionwas centrifuged at 10 000 rpm10min anddried sample analysis was recorded on Perkin Elmer oneIR spectrophotometer in the range from 450 to 3000 cmminus1(Figure 2)The representative spectra in the region of 3000 to

BioMed Research International 3

1080

0893

0707

0521

0335

0148

3000 3800 4600 5400 6200 7000

Abso

rban

ce (a

bs)

WL (nm)

12

(3795 0837)

(4165 0885)1

2(3795 0837)

(4165 0885)

Figure 1 UV-Vis-spectrum of silver nanoparticles

52855

53241

53608

55931

77875

108010116044

122736131348

138645

153860

161381

177102216123

292801

329073

525660

6468

7276808488

92

5001000150020002500300035004000

T(

)

Wavenumbers (cmminus1)

Figure 2 FTIR spectra of silver nanoparticles from A niger

450 cmminus1 revealed the presence of different functional groupslike 329073mdashsecondary amide (NndashH stretch H-bonded)292801mdashalkane (CndashH stretching) 216123mdashalkyne (CequivCstretching) 177102mdashanhydride (C=O stretching) 161381mdashalkene (C=C stretching) 153860mdasharomatic (CndashC stretch-ing) 138645 131348mdash and 108010mdashprimary alcohol (CndashO stretching) and 52855mdashalkene (=CndashH bending) respec-tively Rathod et al [6] reported that proteins present inthe extract of fungus Rhizopus stolonifer can bind to theAgNPs through either free amino or carboxyl groups in theproteins and also they reported different functional groupsabsorbing characteristic frequencies of FTIR radiation Andalso our result correlates with Parashar et al [17] whoreported the possible reaction mechanism for the reductionof Ag+ usingGuara (Psidium guajara) leaf extractThus FTIRis an important and popular tool for structural elucidationand compound identification

A drop of AgNPs solution was placed on the carboncoated copper grids and kept under vacuum before loadingthem onto a specimen holder Then TEM micrographs weretaken by prepared grids to determine the size and shape of theproducedAgNPs Figure 3 revealed the particles are sphericalin shape and size of the particles is between 20 and 55 nmRathod et al [6] synthesized AgNPs from Rhizopus stolonifer

Figure 3 TEMmicrograph of silver nanoparticles synthesized usingextracellular filtrate of A niger

and reported that the size is ranging between 5 and 50 nmsuggesting that biological molecules could possibly performthe function for the stabilization of the AgNPs and alsoreported that the AgNPs synthesized by this route are fairlystable even after prolonged storage Size and shape of AgNPsas revealed by Kora and Arunachalam [15] were 45 nm

The particle size distribution of the AgNPs was shownunder different categories like size distribution by volumeand by intensity The average diameter of the particleswas found to be 73 nm (100 intensity) (Figure 4(a)) witha zeta potential that was minus24mV (Figure 4(b)) The syn-thesized AgNps are well distributed with respect to vol-ume and intensity indicates the well dispersion of AgNPsThe average diameter of the particles was found to be127 100 intensity and width was found to be 3725 nmshowing monodispersity of the particles using the extractof the plant Foeniculum vulgare as reported by Bonde[18]

Scanning electron microscopy (SEM) was used to recordthe photomicrograph images of synthesized AgNPs A smallvolume of AgNPs suspension was taken for SEM analysis onelectromicroscope stub The stubs were placed briefly in adrier and then coated with gold in an ion sputter Pictureswere taken by random scanning of the stub Shape and surfacemorphology of AgNPs were studied by SEM Figure 5(a)shows the distribution of AgNPs SEM micrograph of singlenanoparticle reveals the spherical and smoothmorphology ofthe nanoparticle (Figure 5(b))

Energy dispersive spectroscopy (EDS) samples were pre-pared on a copper substrate by drop coating of AgNPs Theelemental analysis was examined by EDS The presence ofan optical absorption band at sim3 eV reveals the presenceof pure metallic AgNPs along with the C and O signaturesthat might be from the stabilizing protein (Figure 5) Asour result correlates with Li et al [9] who reported theoptical absorption peak at 3 kev from commercially availableAgNPs EDS analysis gives the additional evidence for thereduction of AgNPs the optical absorption peak at 3 kevreported by Parashar et al [17] is typical for the absorptionof metallic AgNPs due to surface Plasmon resonance whichconfirms the presence of nanocrystalline elemental silver (seeFigure 6)

4 BioMed Research International

12

10

8

6

4

2

001 1 10 100 1000 10000

Size distribution by intensity

Inte

nsity

()

Record 10 sample-02 1

Size (dmiddotnm)

(a)

200000

150000

100000

50000

0

minus100 0 100 200

Zeta potential distribution

Apparent zeta potential (mV)

Tota

l cou

nts

Record 10 sample-02 1

(b)

Figure 4 (a) Particle size distribution of AgNPs by intensity with Zeta analyzer (b) Zeta potential

(a) (b)

Figure 5 (a) SEM micrograph of silver nanoparticles (b) single nanoparticle with spherical and smooth morphology

CKa

OKa

NaK

a

AuM

zAu

Ma

AuM

r

AgL

aA

gLb AgL

b2 CaKa

CaKb

AuL1 Au

La

1000

900

800

700

600

500

400

300

200

0

000 100 200 300 400 500 600 700 800 900 1000

Cou

nts

100

C1Kb

C1Ka

(keV)

Figure 6 EDS spectrumof extracellular synthesized silver nanopar-ticles

32 Bactericidal Activities of AgNPs The bactericidal activitywas performed against Gram negative E coli on NA platescontaining different concentrations of AgNPs such as 0 255 10 and 15 120583gmL (Figure 7) Number of bacterial coloniesgrew on NA plate as a function of concentration of AgNPs

when overnight E coli culture was applied to the plates Thepresence of AgNPs at concentrations of 10 and 15120583gmLinhibited bacterial growth by 100 There was luxuriousgrowth at 0120583gmL that is without AgNPs while at 25 120583gmLof AgNPs there was substantial growth maybe because theconcentration of 25120583gmL of AgNPs is not sufficient to killE coli while at 5 120583gmL there was decreased growth dueto enhanced AgNPs concentration Our results indicate that10 120583gmL of AgNPs are sufficient to inhibit 107 CFUmL of Ecoli For confirmation we went for 15 120583gmL concentration ofAgNPs againstE coliHere also therewas complete inhibitionthat indicates that a concentration of 10120583gmL of AgNPs issufficient to completely kill 107 CFUmL of E coli

Li et al [9] also reported that 10 120583gmL of AgNPs aresufficient to completely inhibit 107 CFUmL of E coli thoughthe AgNPs were not biologically synthesized but purchasedcommercially yet our results correlate with them Sondiand Salopek-Sondi [13] also reported that a concentrationof 20120583gmL cmminus3 completely prevented bacterial growth if104 CFU of E coli were used While Kim et al [14] reporteda MIC of 100 120583gmL of AgNPs to cause complete death of Saureus and E coli Comparing our results with the above saidby authors AgNPs produced by A niger are more effectivein completely inhibiting E coli at the minimum dose of

BioMed Research International 5

5120583gmL 15120583gmL

0120583gmL

25 120583gmL

10120583gmL

Figure 7 Bactericidal activities of AgNPs on E coli at different concentrations ranging from 0 120583gmL to 15120583gmL

25

2

15

1

05

0

minus05

5 10 15 20 25

Time (h)

0120583gmL

5120583gmL25 120583gmL

10120583gmL15120583gmL

Abso

rban

ce

Figure 8 Growth curve of E coli treated with AgNPs at 0 120583gmLmaximum growth at 25 120583gmL growth reduced at 5120583gmL bac-terial growth still reduced and at 10 and 15 120583gmL growth curvesdiminished showing nil growth

10 120583gmL Hiremath et al [19] also reported antibacterialactivity ofAgNPs againstMDRE coli strains using the fungusRhizopus sp

33 Growth Curves of Bacterial Cells Treated with AgNPs Thegrowth curve of E coli treated with AgNPs was determinedby using NB supplemented with 0 25 5 10 and 15120583gmLof AgNPs The concentrations of 25 and 5 120583gmL showedthe growth of E coli but comparatively less than the growthcurve shown without AgNPs (0120583gmL) It is very interestingto observe that the incorporations of 10 and 15 120583gmL AgNPsto NB showed complete inhibition of E coli which is evidentfrom graph in Figure 8 The dynamics of bacterial growthwas monitored in liquid LB broth supplemented with 107 Ecoli cells with 10 50 and 100 120583g cmminus3 of AgNPs at all theseconcentrations caused a growth delay of E coli Zhou et al[20] also reported that a concentration of 10 120583gmL of AgNPsshowed strong antibacterial activity against E coli

34 Release of ROS from the Surface of AgNPs and ItsMechanism Gram negative E coli was selected as a model

6 BioMed Research International

250nm

(a)

200nm

(b)

200nm

(c)

200nm

(d)

150nm

(e)

200nm

(f)

Figure 9 TEMmicrograph of E coli loaded with AgNPs (a) Normal E coli cell with its well-integrated cell wall (b) Anchoring of AgNPs oncell wall of E coli (c) Ruptured cell membrane and entry of AgNPs into the cytoplasm (d) Complete damage of cell wall and cell membrane(e) Clear AgNPs within the E coli cell (f) Complete destruction of E coli cell and overloading of AgNPs

to study the effect of ROS released from the surface of AgNPson the permeability and the membrane structure of E colicells The interaction of AgNPs with bacterium was analyzedusing TEMmicrographs E coli samples were incubated withAgNPs for 12 hours and were analyzed by employing TEMFigure 9(a) shows normal E coli cells with its well-integratedcell wall When the E coli cells were made to interact withAgNPs for 1 hr it can be seen fromFigure 9(b) that theAgNPswere trying to adhere to the surface of E coli cells After 5 hrsof interaction a closure look at the bacterial cell membranereveals that AgNPs anchored onto the cell surface of E coliandmany pits and gaps appeared in themicrograph and theirmembrane was fragmented (Figure 9(c)) Figure 9(d) shows

that AgNPs are trying to get inside the bacterial cell throughthe ruptured cell membrane After 8 hours the AgNPssurround the bacterial cell surface and almost cover the sidesof the cell and nanoparticles are visible in the cytoplasm alsoIn addition electron dense particles or precipitates were alsoobserved around the damaged bacterial cell (Figure 9(e))The interaction can either completely disintegrates the cell ormay cause cell lyses after 12 hours Figure 9(f) shows AgNPseventually leading to the bacterial cell death

Another proposed mechanism of E colimembrane dam-age byAgNPs relates tometal depletion that is the formationof pits in the outer membrane and change in membranepermeability by the progressive release of lipopolysaccharide

BioMed Research International 7

2047

minus2048

327640 (mT)

(A) 337475 g = 200025

337640 (mT) 347640 (mT)

(m1) 332715 g = 202887(m2 minus m1) = 87037 (mT)

(m2) 341418 g = 197715

Figure 10 ESR spectrum of AgNPs recorded at room temperaturem1

(332715) and m2

(341418) indicates the control peak of Mnand the peak (mT 337475) indicates the released free radical fromAgNPs Instrument setting JEOL-JES-TE 200 spectrophotometermicropower 4mW MOD 100 khz and the time const 003 sec

20E+06

15E+06

10E+06

50E+05

0

500 600 700 800

Wavelength (nm)

Emission acquistion

Inte

nsity

(cps

)

File 1 = VRSRJ01

Figure 11 Formation of ROS in E coli using fluorescence spec-troscopy

(LPS) molecules and membrane proteins A bacterial mem-brane with this morphology exhibits a significant increase inpermeability leaving the bacterial cells incapable of properlyregulating transport through the plasma membrane andfinally causing cell death It is well known that the outermembrane of E coli cells is predominantly constructed fromtightly packed LPS molecules which provide an effectivepermeability barrier [13] It has been also proposed that thesites of interaction for AgNPs and membrane cells might bedue to sulfur containing proteins in a similar way as silverinteracts with thiol groups of respiratory chain proteins andtransport proteins interfering with their proper function [21ndash25]

Reactive Oxygen Species (ROS) are natural byproducts ofthemetabolismof respiring organisms [16] Induction of ROSsynthesis leads to the formation of highly reactive radicalsthat destroy the cells The bacterial growth inhibition byformation of free radicals from the surface of AgNPs was alsoobserved by employing ESR spectroscopy (Figure 10) Excessgeneration of reactive oxygen species can attack membranelipids and then lead to a breakdown of membrane functionCertain transition metals might disrupt the cellular donorligands that coordinate Fe Mounting evidence suggests that

the primary targets for variousmetals are the solvent exposed[4Fe-4S] clusters of proteins The direct or indirect destruc-tion of [4Fe-4S] clusters by metals could result in the releaseof additional Fenton-active Fe into the cytoplasm resultingin increased ROS formation The ability to induce Fe releasefrom these proteins as well as from other Fe-containingproteins might account for observations that some Fenton-inactive metals (such as Ag Hg and Ga) generate ROS andthat cells require or upregulate ROS-detoxification enzymesto withstand toxic doses of these nanoparticles [26]The ROSproduction from the surface of AgNPs was measured usingDCFH-DAmethod Dichlorofluorescein diacetate (DCFDA)is a popular fluorescence-based probe for reactive oxygenspecies (ROS) detection in vitro After 5 h of incubationROS formed in the sample was detected at 523 nm of emis-sion wavelength using Fluorescence spectroscopy (Figure 11)Intracellular esterases cleave DCFH-DA at the two esterbonds producing a relatively polar and cell membrane-impermeable product H2DCFThis nonfluorescentmoleculeaccumulates intracellularly and subsequent oxidation yieldsthe highly fluorescent product DCF The redox state of thesample can bemonitored by detecting the increase in fluores-cence The result confirms the generation of free radicals andfrom the surface of AgNPs which becomes toxic to bacterialcells leading to death Our result corroborates with Kim etal [14] who reported the oxidative stress can cause damageto bacterial cell membrane protein structure and intracel-luluar system against S aureus and E coli usingDCFDAmethod

To determine the involvement of ROS in the antibacterialactivity of AgNPs we used ascorbic acid as scavenger Thisantioxidant was used to scavenge the ROS produced bythe AgNPs Protective activity of antioxidant against bac-tericidal activity of AgNPs was observed in Figure 12 Incontrol plate the bacterial colonies were clearly seen withoutantioxidants and AgNPs but in the plate supplementedwith AgNPs (10 120583gmL) no bacterial growth was observedrevealing that AgNPs completely inhibited bacterial growthdue to ROS formation Surprisingly the bacterial colonieswere observed in the plate supplemented with both AgNPsand antioxidant which clearly indicates that the ascorbicacid used as an antioxidant serves as a scavenger hinderingthe ROS release from AgNPs It is determined that theantioxidant prevents the formation of a silver oxide layeron the AgNPs surface and consequently formation of theAg+ reservoir Kora and Arunachalam [15] reported the100 P aeruginosa growth survival by using ascorbic acidas scavenger while 73 of bacteria protected from NAC asscavenger

Bacterial cells exposed to AgNPs suffer morphologicalchanges such as cytoplasm shrinkage detachment of cellwall membrane DNA condensation and localization in anelectron-light region in the centre of the cell and cellmembrane degradation allowing the leakage of intracellularcontents [14 27] Physiological changes occur together withthe morphological changes bacterial cells enter an activebut nonculturable state in which physiological levels can bemeasured but cells are not able to grow and replicate [9 28]

8 BioMed Research International

Control SNPs SNPs + ascorbic acid

Figure 12 Antioxidant activities of silver nanoparticles on E coli

4 Conclusion

From our results two aspects can be elucidated one is thefact that AgNPs themselves having antibacterial propertiesact on the pathogenic bacteria by anchoring to the cell surfacetrying to get inside the bacterial cell through ruptured cellmembrane leading to the perforation of cell membrane bycausing leakage of cell components and finally cell deathThesecond aspect is the release of ROS from AgNPs producedby A niger which is clearly evident from the facts thatthe antioxidant ascorbic acid is used as a scavenger ROSproductionwas confirmed by usingDCFH-DAmethodusingfluorescence spectroscopy Antioxidant will inhibit ROS pro-duction thereby luxurious growth of E coli was observedNutrient agar plate inoculated with E coli supplemented withAgNPs without addition of antioxidant showed completeinhibition of E coli suggesting that ROS is released fromAgNPs which completely inhibit E coli Our studies suggestthat 10 120583gmL of AgNPs are sufficient for complete inhibitionof E coli which is a boon to biotechnologists to go deep formechanism studies

Conflict of Interests

The authors report no conflict of interests

Acknowledgments

The Authors would like to thank the Department of Micro-biology Gulbarga University Gulbarga India for provid-ing logistic support and facilities They also thank SAIF-STIC Cochin Ruska labs Hyderabad SAIF-IIT Madras andMalvern Instruments Ltd Bangalore India for characteri-zation studies

References

[1] CMarambio-Jones and EM VHoek ldquoA review of the antibac-terial effects of silver nanomaterials and potential implicationsfor human health and the environmentrdquo Journal of NanoparticleResearch vol 12 no 5 pp 1531ndash1551 2010

[2] D Rejeski ldquoNanotechnology and consumer productsrdquohttpwwwnanotechprojectorgpublicationsarchivenano-technology consumer products

[3] D Singh V Rathod N Shivaraj J Hiremath A K Singhand J Methew ldquoOptimization and characterization of silvernanoparticles by endophytic fungi Penicillium sp isolated from

Curcuma longa (turmeric) and Application studies againstMDR E coli and S aureusrdquo Bioinorganic Chemistry and Appli-cations vol 2014 Article ID 408021 8 pages 2014

[4] A Banu V Rathod and E Ranganath ldquoSilver nanoparticleproduction by Rhizopus stolonifer and its antibacterial activ-ity against extended spectrum 120573-lactamase producing (ESBL)strains of Enterobacteriaceaerdquo Materials Research Bulletin vol46 no 9 pp 1417ndash1423 2011

[5] D Singh V Rathod N Shivaraj J Hiremath and P Kulka-rni ldquoBiosynthesis of silver nanoparticles by endophytic fungiPenicillium sp isolated from Curcuma longa (turmeric) and itsantibacterial activity against pathogenic gram negative bacte-riardquo Journal of Pharmacy Research vol 7 no 5 pp 448ndash4532013

[6] V Rathod A Banu and E Ranganath ldquoBiosynthesis of highlystabilized silver nanoparticles by Rhizopus stolonifer and theirAnti-fungal efficacyrdquo International Journal of Molecular andClinical Microbiology vol 1 no 2 pp 65ndash70 2011

[7] S Galdiero A Falanga M Vitiello M Cantisani V Marra andM Galdiero ldquoSilver nanoparticles as potential antiviral agentsrdquoMolecules vol 16 no 10 pp 8894ndash8918 2011

[8] Y Wang L Cao S Guan et al ldquoSilver mineralization onself-assembled peptide nanofibers for long term antimicrobialeffectrdquo Journal of Materials Chemistry vol 22 no 6 pp 2575ndash2581 2012

[9] W-R Li X-B Xie Q-S Shi H-Y Zeng Y-S Ou-Yang andY-B Chen ldquoAntibacterial activity and mechanism of silvernanoparticles on Escherichia colirdquo Applied Microbiology andBiotechnology vol 85 no 4 pp 1115ndash1122 2010

[10] J M Galloway and S S Staniland ldquoProtein and peptidebiotemplated metal and metal oxide nanoparticles and theirpatterning onto surfacesrdquo Journal of Materials Chemistry vol22 no 25 pp 12423ndash12434 2012

[11] P Mohanpuria N K Rana and S K Yadav ldquoBiosynthesis ofnanoparticles technological concepts and future applicationsrdquoJournal of Nanoparticle Research vol 10 no 3 pp 507ndash517 2008

[12] S K Das and E Marsili ldquoA green chemical approach for thesynthesis of gold nanoparticles characterization andmechanis-tic aspectrdquo Reviews in Environmental Science and Biotechnologyvol 9 no 3 pp 199ndash204 2010

[13] I Sondi and B Salopek-Sondi ldquoSilver nanoparticles as antimi-crobial agent a case study on E coli as a model for Gram-negative bacteriardquo Journal of Colloid and Interface Science vol275 no 1 pp 177ndash182 2004

[14] S-H Kim H-S Lee D-S Ryu S-J Choi and D-S LeeldquoAntibacterial activity of silver-nanoparticles against Staphylo-coccus aureus and Escherichia colirdquo Korean Journal of Microbiol-ogy and Biotechnology vol 39 no 1 pp 77ndash85 2011

BioMed Research International 9

[15] A J Kora and J Arunachalam ldquoAssessment of antibacterialactivity of silver nanoparticles on Pseudomonas aeruginosa andits mechanism of actionrdquo World Journal of Microbiology andBiotechnology vol 27 no 5 pp 1209ndash1216 2011

[16] J S Kim E Kuk K N Yu et al ldquoAntimicrobial effects of silvernanoparticlesrdquo Nanomedicine vol 3 no 1 pp 95ndash101 2007

[17] U K Parashar V Kumar T Bera et al ldquoStudy of mechanismof enhanced antibacterial activity by green synthesis of silvernanoparticlesrdquo Nanotechnology vol 22 no 41 Article ID415104 13 pages 2011

[18] S Bonde ldquoA Biogenic approach for green synthesis of silvernanoparticles using extract of Foeniculum vulgare and itsactivity against S aureus and Ecolirdquo Bioscience vol 3 no 1 pp59ndash63 2011

[19] J Hiremath V Rathod S Ninganagouda D Singh and KPrema ldquoAntibacterial activity of Silver Nanoparticles from Rhi-zopus spp against Gram negative EcoliMDR strainsrdquo Journal ofPure and Applied Microbiology vol 8 no 1 pp 555ndash562 2014

[20] Y Zhou Y Kong S Kundu J D Cirillo and H LiangldquoAntibacterial activities of gold and silver nanoparticles againstEscherichia coli and bacillus Calmette-Guerinrdquo Journal ofNanobiotechnology vol 10 article 19 2012

[21] W K Jung H C Koo K W Kim S Shin S H Kim and YH Park ldquoAntibacterial activity and mechanism of action of thesilver ion in Staphylococcus aureus and Escherichia colirdquoAppliedand Environmental Microbiology vol 74 no 7 pp 2171ndash21782008

[22] M Yamanaka K Hara and J Kudo ldquoBactericidal actions ofa silver ion solution on Escherichia coli studied by energy-filtering transmission electron microscopy and proteomic anal-ysisrdquoApplied and EnvironmentalMicrobiology vol 71 no 11 pp7589ndash7593 2005

[23] Q L Feng J Wu G Q Chen T N Kim and J O Kim ldquoAmechanistic study of the antibacterial effect of silver ions onEscherichia coli and Staphylococcus aureusrdquo Journal of Biomedi-cal Materials Research vol 52 no 4 pp 662ndash668 2000

[24] J R Morones J L Elechiguerra A Camacho et al ldquoThebactericidal effect of silver nanoparticlesrdquo Nanotechnology vol16 no 10 pp 2346ndash2353 2005

[25] Y Matsumura K Yoshikata S-I Kunisaki and T TsuchidoldquoMode of bactericidal action of silver zeolite and its comparisonwith that of silver nitraterdquo Applied and Environmental Microbi-ology vol 69 no 7 pp 4278ndash4281 2003

[26] J A Lemire J JHarrison andR J Turner ldquoAntimicrobial activ-ity of metals mechanisms molecular targets and applicationsrdquoNature Reviews Microbiology vol 11 no 6 pp 371ndash384 2013

[27] AGrigorEva I Saranina N Tikunova et al ldquoFinemechanismsof the interaction of silver nanoparticles with the cells ofSalmonella typhimurium and Staphylococcus aureusrdquo BioMetalsvol 26 no 3 pp 479ndash488 2013

[28] M Chen Z Yang H Wu X Pan X Xie and C WuldquoAntimicrobial activity and the mechanism of silver nanoparti-cle thermosensitive gelrdquo International Journal of Nanomedicinevol 6 pp 2873ndash2877 2011

Submit your manuscripts athttpwwwhindawicom

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MEDIATORSINFLAMMATION

of

Page 2: Research Article Growth Kinetics and Mechanistic Action of ...downloads.hindawi.com/journals/bmri/2014/753419.pdf · Species Released by Silver Nanoparticles from Aspergillus niger

2 BioMed Research International

Escherichia coli and confirm the obtained transmission elec-tron microscopy (TEM) results by Kinetic studies throughgrowth curve

2 Materials and Methods

21 Biosynthesis and Characterization of AgNPs The fungusA niger was grown in 250mL Erlenmeyer flasks containingmalt extract glucose yeast extract and peptone (MGYP-03 1 03 and 05 and pH 6) at temperature 29∘CAfter incubation mycelium was separated by filtrationwashed with sterile distilled water to remove traces of mediacontents and resuspended in 100mL distilled water for about48 hrs The suspension was filtered through Whatman filterpaper number 1 The cell filtrate was challenged with silvernitrate (1mM) for AgNPs biosynthesis

The Optical density of the synthesized AgNPs was char-acterized by UV-Vis spectroscopy (T 90+ UV-VIS spec-trophotometer) the interaction between protein and AgNPswas evaluated using Fourier-transform infrared spectroscopy(FTIR) (Perkin Elmer model 783 spectrophotometer) thesize and morphology of the AgNPs were examined bytransmission electron microscopy (TEM) (Hitachi H 7500ID Japan) the particle size distribution of AgNPs wasevaluated using Malvern Zetasizer nanoseries spectrometer(Malvern Instruments Ltd) shape and surface morphologyof nanoparticleswas characterized by using scanning electronmicroscopy (SEM) (JEOLModel JSM-6390 LV) the presenceof elemental silver was confirmed through energy dispersivespectroscopy (EDS) (JEOL Model JED-2300) and release ofreactive oxygen species (ROS) from the surface of AgNPs wasevaluated by employing electron spin resonance spectroscopy(ESR) (JES-FA 200ESR Spectrometer JEOL Japan)

22 Bactericidal Activity of AgNPs Agar dilution methodwas used to study the bactericidal activity of AgNPs onE coli Nutrient Agar (NA) supplemented with variousconcentrations ofAgNPs (0 25 5 10 and 15120583gmL) and eachplate was inoculated with 107 CFUmL of E coli by spreadplating Plates inoculatedwith E coli but without AgNPswereused as control Number of surviving bacteria in agar plateswas counted after 24 hours of incubation at 37∘C [13 14]

23 Bacterial GrowthKinetics against Different Concentrationsof AgNPs To study the bacterial growth curve E coli culturewas inoculated with fresh colonies and incubated for 12 hourovernight at 37∘C in nutrient broth (NB) Bacterial growthcurves were determined by measuring the optical density(OD) at 600 nm using a spectrophotometer At this thejuncture theODobtainedwas 10 To different concentrations(0ndash15120583gmL) ofAgNPs the aforesaidE coli culturewas addedand the turbidity was measured at different time intervals (05 10 15 20 and 25 hrs) [14] Experiment was repeated thrice

24 Release of ROS from the Surface of AgNPs and Its Mech-anism The interaction of AgNPs with E coli was assessedusing TEM Broth containing E coli exposed to AgNPswas subjected to TEM at a time intervals of 1 5 8 and

12 hrs respectively The samples which were subjected toTEM studies were also sent for ESR studies to detect thefree radical generation from the surface of silver ESR is ananalytical method to detect the free radical generated fromthe surface of silver It is based on absorption of microwaveradiation by an unpaired electron when it is exposed toa strong magnetic field AgNPs that contain free radicalstherefore are detected by ESR Free radical generation fromthe surface of Agwas recorded using ESR spectrophotometerThe generation of ROS from AgNPs was measured using2101584071015840-dichlorofluorescein diacetate (DCFH-DA) [14] 2101584071015840-Dichlorodihydrofluorescein diacetate (DCFH-DA) is one ofthe most widely used techniques for directly measuring theredox state of a cell DCFH-DA a cell permeable nonfluores-cent precursor of DCF can be used as an intracellular probefor oxidative stress It has many advantages over other tech-niques developed as it is very easy to use extremely sensitiveto changes in the redox state of a cell inexpensive and canbe used to follow changes in ROS over time 107 CFUmL ofcells were treated with 20120583gmL of AgNPs and incubated at37∘C for 5 h then centrifuged at 4∘C for 15min at 600timesg andthe obtained supernatantwas treatedwith 100120583MDCFH-DAfor 1 h The ROS formed was measured using Fluorescencespectrometry

To check the inhibition of E coli targeted through theROS produced by AgNPs from A niger we conducted a sep-arate experiment using ascorbic acid as an antioxidant whichacts as a scavenger NA plates supplemented with AgNPs(10 120583gmL) were also incorporated with 10mM ascorbic acidas a scavenger Thus the plates were inoculated with fresh12 hrs cultures of E coli and the surviving rate was countedafter 24 hrs of incubation [15ndash17]

3 Results and Discussion

31 Biosynthesis and Characterization of AgNPs The produc-tion of AgNPs by A niger was indicated by the appearance ofbrown color in the reaction mixture The UV-Vis spectrumfor AgNPs is obtained by exposing the sample to UV-lightfrom a light sourceThe specific surface plasmon resonance isresponsible for their unique remarkable optical phenomenonA single peak with a maximum of 416 nm corresponding tothe surface plasmon resonance of AgNPs was observed inthe UV-Vis spectrum (Figure 1) Singh et al [3] reported theextracellular biosynthesis of AgNPs using endophytic fungusPenicillium sp at the maximum absorbance peak of 425 nmand also reported that increase in concentration of silvernitrate increases the particle size due to aggregation of largerAgNPs In an another study Sondi and Salopek-Sondi [13]reported the surface Plasmon band at 405 nm at this peak theappearance of brown color clearly indicates the formation ofAgNPs

The interaction between protein andAgNPswas analyzedby Fourier-transform infrared spectroscopy (FTIR) TheAgNPs suspensionwas centrifuged at 10 000 rpm10min anddried sample analysis was recorded on Perkin Elmer oneIR spectrophotometer in the range from 450 to 3000 cmminus1(Figure 2)The representative spectra in the region of 3000 to

BioMed Research International 3

1080

0893

0707

0521

0335

0148

3000 3800 4600 5400 6200 7000

Abso

rban

ce (a

bs)

WL (nm)

12

(3795 0837)

(4165 0885)1

2(3795 0837)

(4165 0885)

Figure 1 UV-Vis-spectrum of silver nanoparticles

52855

53241

53608

55931

77875

108010116044

122736131348

138645

153860

161381

177102216123

292801

329073

525660

6468

7276808488

92

5001000150020002500300035004000

T(

)

Wavenumbers (cmminus1)

Figure 2 FTIR spectra of silver nanoparticles from A niger

450 cmminus1 revealed the presence of different functional groupslike 329073mdashsecondary amide (NndashH stretch H-bonded)292801mdashalkane (CndashH stretching) 216123mdashalkyne (CequivCstretching) 177102mdashanhydride (C=O stretching) 161381mdashalkene (C=C stretching) 153860mdasharomatic (CndashC stretch-ing) 138645 131348mdash and 108010mdashprimary alcohol (CndashO stretching) and 52855mdashalkene (=CndashH bending) respec-tively Rathod et al [6] reported that proteins present inthe extract of fungus Rhizopus stolonifer can bind to theAgNPs through either free amino or carboxyl groups in theproteins and also they reported different functional groupsabsorbing characteristic frequencies of FTIR radiation Andalso our result correlates with Parashar et al [17] whoreported the possible reaction mechanism for the reductionof Ag+ usingGuara (Psidium guajara) leaf extractThus FTIRis an important and popular tool for structural elucidationand compound identification

A drop of AgNPs solution was placed on the carboncoated copper grids and kept under vacuum before loadingthem onto a specimen holder Then TEM micrographs weretaken by prepared grids to determine the size and shape of theproducedAgNPs Figure 3 revealed the particles are sphericalin shape and size of the particles is between 20 and 55 nmRathod et al [6] synthesized AgNPs from Rhizopus stolonifer

Figure 3 TEMmicrograph of silver nanoparticles synthesized usingextracellular filtrate of A niger

and reported that the size is ranging between 5 and 50 nmsuggesting that biological molecules could possibly performthe function for the stabilization of the AgNPs and alsoreported that the AgNPs synthesized by this route are fairlystable even after prolonged storage Size and shape of AgNPsas revealed by Kora and Arunachalam [15] were 45 nm

The particle size distribution of the AgNPs was shownunder different categories like size distribution by volumeand by intensity The average diameter of the particleswas found to be 73 nm (100 intensity) (Figure 4(a)) witha zeta potential that was minus24mV (Figure 4(b)) The syn-thesized AgNps are well distributed with respect to vol-ume and intensity indicates the well dispersion of AgNPsThe average diameter of the particles was found to be127 100 intensity and width was found to be 3725 nmshowing monodispersity of the particles using the extractof the plant Foeniculum vulgare as reported by Bonde[18]

Scanning electron microscopy (SEM) was used to recordthe photomicrograph images of synthesized AgNPs A smallvolume of AgNPs suspension was taken for SEM analysis onelectromicroscope stub The stubs were placed briefly in adrier and then coated with gold in an ion sputter Pictureswere taken by random scanning of the stub Shape and surfacemorphology of AgNPs were studied by SEM Figure 5(a)shows the distribution of AgNPs SEM micrograph of singlenanoparticle reveals the spherical and smoothmorphology ofthe nanoparticle (Figure 5(b))

Energy dispersive spectroscopy (EDS) samples were pre-pared on a copper substrate by drop coating of AgNPs Theelemental analysis was examined by EDS The presence ofan optical absorption band at sim3 eV reveals the presenceof pure metallic AgNPs along with the C and O signaturesthat might be from the stabilizing protein (Figure 5) Asour result correlates with Li et al [9] who reported theoptical absorption peak at 3 kev from commercially availableAgNPs EDS analysis gives the additional evidence for thereduction of AgNPs the optical absorption peak at 3 kevreported by Parashar et al [17] is typical for the absorptionof metallic AgNPs due to surface Plasmon resonance whichconfirms the presence of nanocrystalline elemental silver (seeFigure 6)

4 BioMed Research International

12

10

8

6

4

2

001 1 10 100 1000 10000

Size distribution by intensity

Inte

nsity

()

Record 10 sample-02 1

Size (dmiddotnm)

(a)

200000

150000

100000

50000

0

minus100 0 100 200

Zeta potential distribution

Apparent zeta potential (mV)

Tota

l cou

nts

Record 10 sample-02 1

(b)

Figure 4 (a) Particle size distribution of AgNPs by intensity with Zeta analyzer (b) Zeta potential

(a) (b)

Figure 5 (a) SEM micrograph of silver nanoparticles (b) single nanoparticle with spherical and smooth morphology

CKa

OKa

NaK

a

AuM

zAu

Ma

AuM

r

AgL

aA

gLb AgL

b2 CaKa

CaKb

AuL1 Au

La

1000

900

800

700

600

500

400

300

200

0

000 100 200 300 400 500 600 700 800 900 1000

Cou

nts

100

C1Kb

C1Ka

(keV)

Figure 6 EDS spectrumof extracellular synthesized silver nanopar-ticles

32 Bactericidal Activities of AgNPs The bactericidal activitywas performed against Gram negative E coli on NA platescontaining different concentrations of AgNPs such as 0 255 10 and 15 120583gmL (Figure 7) Number of bacterial coloniesgrew on NA plate as a function of concentration of AgNPs

when overnight E coli culture was applied to the plates Thepresence of AgNPs at concentrations of 10 and 15120583gmLinhibited bacterial growth by 100 There was luxuriousgrowth at 0120583gmL that is without AgNPs while at 25 120583gmLof AgNPs there was substantial growth maybe because theconcentration of 25120583gmL of AgNPs is not sufficient to killE coli while at 5 120583gmL there was decreased growth dueto enhanced AgNPs concentration Our results indicate that10 120583gmL of AgNPs are sufficient to inhibit 107 CFUmL of Ecoli For confirmation we went for 15 120583gmL concentration ofAgNPs againstE coliHere also therewas complete inhibitionthat indicates that a concentration of 10120583gmL of AgNPs issufficient to completely kill 107 CFUmL of E coli

Li et al [9] also reported that 10 120583gmL of AgNPs aresufficient to completely inhibit 107 CFUmL of E coli thoughthe AgNPs were not biologically synthesized but purchasedcommercially yet our results correlate with them Sondiand Salopek-Sondi [13] also reported that a concentrationof 20120583gmL cmminus3 completely prevented bacterial growth if104 CFU of E coli were used While Kim et al [14] reporteda MIC of 100 120583gmL of AgNPs to cause complete death of Saureus and E coli Comparing our results with the above saidby authors AgNPs produced by A niger are more effectivein completely inhibiting E coli at the minimum dose of

BioMed Research International 5

5120583gmL 15120583gmL

0120583gmL

25 120583gmL

10120583gmL

Figure 7 Bactericidal activities of AgNPs on E coli at different concentrations ranging from 0 120583gmL to 15120583gmL

25

2

15

1

05

0

minus05

5 10 15 20 25

Time (h)

0120583gmL

5120583gmL25 120583gmL

10120583gmL15120583gmL

Abso

rban

ce

Figure 8 Growth curve of E coli treated with AgNPs at 0 120583gmLmaximum growth at 25 120583gmL growth reduced at 5120583gmL bac-terial growth still reduced and at 10 and 15 120583gmL growth curvesdiminished showing nil growth

10 120583gmL Hiremath et al [19] also reported antibacterialactivity ofAgNPs againstMDRE coli strains using the fungusRhizopus sp

33 Growth Curves of Bacterial Cells Treated with AgNPs Thegrowth curve of E coli treated with AgNPs was determinedby using NB supplemented with 0 25 5 10 and 15120583gmLof AgNPs The concentrations of 25 and 5 120583gmL showedthe growth of E coli but comparatively less than the growthcurve shown without AgNPs (0120583gmL) It is very interestingto observe that the incorporations of 10 and 15 120583gmL AgNPsto NB showed complete inhibition of E coli which is evidentfrom graph in Figure 8 The dynamics of bacterial growthwas monitored in liquid LB broth supplemented with 107 Ecoli cells with 10 50 and 100 120583g cmminus3 of AgNPs at all theseconcentrations caused a growth delay of E coli Zhou et al[20] also reported that a concentration of 10 120583gmL of AgNPsshowed strong antibacterial activity against E coli

34 Release of ROS from the Surface of AgNPs and ItsMechanism Gram negative E coli was selected as a model

6 BioMed Research International

250nm

(a)

200nm

(b)

200nm

(c)

200nm

(d)

150nm

(e)

200nm

(f)

Figure 9 TEMmicrograph of E coli loaded with AgNPs (a) Normal E coli cell with its well-integrated cell wall (b) Anchoring of AgNPs oncell wall of E coli (c) Ruptured cell membrane and entry of AgNPs into the cytoplasm (d) Complete damage of cell wall and cell membrane(e) Clear AgNPs within the E coli cell (f) Complete destruction of E coli cell and overloading of AgNPs

to study the effect of ROS released from the surface of AgNPson the permeability and the membrane structure of E colicells The interaction of AgNPs with bacterium was analyzedusing TEMmicrographs E coli samples were incubated withAgNPs for 12 hours and were analyzed by employing TEMFigure 9(a) shows normal E coli cells with its well-integratedcell wall When the E coli cells were made to interact withAgNPs for 1 hr it can be seen fromFigure 9(b) that theAgNPswere trying to adhere to the surface of E coli cells After 5 hrsof interaction a closure look at the bacterial cell membranereveals that AgNPs anchored onto the cell surface of E coliandmany pits and gaps appeared in themicrograph and theirmembrane was fragmented (Figure 9(c)) Figure 9(d) shows

that AgNPs are trying to get inside the bacterial cell throughthe ruptured cell membrane After 8 hours the AgNPssurround the bacterial cell surface and almost cover the sidesof the cell and nanoparticles are visible in the cytoplasm alsoIn addition electron dense particles or precipitates were alsoobserved around the damaged bacterial cell (Figure 9(e))The interaction can either completely disintegrates the cell ormay cause cell lyses after 12 hours Figure 9(f) shows AgNPseventually leading to the bacterial cell death

Another proposed mechanism of E colimembrane dam-age byAgNPs relates tometal depletion that is the formationof pits in the outer membrane and change in membranepermeability by the progressive release of lipopolysaccharide

BioMed Research International 7

2047

minus2048

327640 (mT)

(A) 337475 g = 200025

337640 (mT) 347640 (mT)

(m1) 332715 g = 202887(m2 minus m1) = 87037 (mT)

(m2) 341418 g = 197715

Figure 10 ESR spectrum of AgNPs recorded at room temperaturem1

(332715) and m2

(341418) indicates the control peak of Mnand the peak (mT 337475) indicates the released free radical fromAgNPs Instrument setting JEOL-JES-TE 200 spectrophotometermicropower 4mW MOD 100 khz and the time const 003 sec

20E+06

15E+06

10E+06

50E+05

0

500 600 700 800

Wavelength (nm)

Emission acquistion

Inte

nsity

(cps

)

File 1 = VRSRJ01

Figure 11 Formation of ROS in E coli using fluorescence spec-troscopy

(LPS) molecules and membrane proteins A bacterial mem-brane with this morphology exhibits a significant increase inpermeability leaving the bacterial cells incapable of properlyregulating transport through the plasma membrane andfinally causing cell death It is well known that the outermembrane of E coli cells is predominantly constructed fromtightly packed LPS molecules which provide an effectivepermeability barrier [13] It has been also proposed that thesites of interaction for AgNPs and membrane cells might bedue to sulfur containing proteins in a similar way as silverinteracts with thiol groups of respiratory chain proteins andtransport proteins interfering with their proper function [21ndash25]

Reactive Oxygen Species (ROS) are natural byproducts ofthemetabolismof respiring organisms [16] Induction of ROSsynthesis leads to the formation of highly reactive radicalsthat destroy the cells The bacterial growth inhibition byformation of free radicals from the surface of AgNPs was alsoobserved by employing ESR spectroscopy (Figure 10) Excessgeneration of reactive oxygen species can attack membranelipids and then lead to a breakdown of membrane functionCertain transition metals might disrupt the cellular donorligands that coordinate Fe Mounting evidence suggests that

the primary targets for variousmetals are the solvent exposed[4Fe-4S] clusters of proteins The direct or indirect destruc-tion of [4Fe-4S] clusters by metals could result in the releaseof additional Fenton-active Fe into the cytoplasm resultingin increased ROS formation The ability to induce Fe releasefrom these proteins as well as from other Fe-containingproteins might account for observations that some Fenton-inactive metals (such as Ag Hg and Ga) generate ROS andthat cells require or upregulate ROS-detoxification enzymesto withstand toxic doses of these nanoparticles [26]The ROSproduction from the surface of AgNPs was measured usingDCFH-DAmethod Dichlorofluorescein diacetate (DCFDA)is a popular fluorescence-based probe for reactive oxygenspecies (ROS) detection in vitro After 5 h of incubationROS formed in the sample was detected at 523 nm of emis-sion wavelength using Fluorescence spectroscopy (Figure 11)Intracellular esterases cleave DCFH-DA at the two esterbonds producing a relatively polar and cell membrane-impermeable product H2DCFThis nonfluorescentmoleculeaccumulates intracellularly and subsequent oxidation yieldsthe highly fluorescent product DCF The redox state of thesample can bemonitored by detecting the increase in fluores-cence The result confirms the generation of free radicals andfrom the surface of AgNPs which becomes toxic to bacterialcells leading to death Our result corroborates with Kim etal [14] who reported the oxidative stress can cause damageto bacterial cell membrane protein structure and intracel-luluar system against S aureus and E coli usingDCFDAmethod

To determine the involvement of ROS in the antibacterialactivity of AgNPs we used ascorbic acid as scavenger Thisantioxidant was used to scavenge the ROS produced bythe AgNPs Protective activity of antioxidant against bac-tericidal activity of AgNPs was observed in Figure 12 Incontrol plate the bacterial colonies were clearly seen withoutantioxidants and AgNPs but in the plate supplementedwith AgNPs (10 120583gmL) no bacterial growth was observedrevealing that AgNPs completely inhibited bacterial growthdue to ROS formation Surprisingly the bacterial colonieswere observed in the plate supplemented with both AgNPsand antioxidant which clearly indicates that the ascorbicacid used as an antioxidant serves as a scavenger hinderingthe ROS release from AgNPs It is determined that theantioxidant prevents the formation of a silver oxide layeron the AgNPs surface and consequently formation of theAg+ reservoir Kora and Arunachalam [15] reported the100 P aeruginosa growth survival by using ascorbic acidas scavenger while 73 of bacteria protected from NAC asscavenger

Bacterial cells exposed to AgNPs suffer morphologicalchanges such as cytoplasm shrinkage detachment of cellwall membrane DNA condensation and localization in anelectron-light region in the centre of the cell and cellmembrane degradation allowing the leakage of intracellularcontents [14 27] Physiological changes occur together withthe morphological changes bacterial cells enter an activebut nonculturable state in which physiological levels can bemeasured but cells are not able to grow and replicate [9 28]

8 BioMed Research International

Control SNPs SNPs + ascorbic acid

Figure 12 Antioxidant activities of silver nanoparticles on E coli

4 Conclusion

From our results two aspects can be elucidated one is thefact that AgNPs themselves having antibacterial propertiesact on the pathogenic bacteria by anchoring to the cell surfacetrying to get inside the bacterial cell through ruptured cellmembrane leading to the perforation of cell membrane bycausing leakage of cell components and finally cell deathThesecond aspect is the release of ROS from AgNPs producedby A niger which is clearly evident from the facts thatthe antioxidant ascorbic acid is used as a scavenger ROSproductionwas confirmed by usingDCFH-DAmethodusingfluorescence spectroscopy Antioxidant will inhibit ROS pro-duction thereby luxurious growth of E coli was observedNutrient agar plate inoculated with E coli supplemented withAgNPs without addition of antioxidant showed completeinhibition of E coli suggesting that ROS is released fromAgNPs which completely inhibit E coli Our studies suggestthat 10 120583gmL of AgNPs are sufficient for complete inhibitionof E coli which is a boon to biotechnologists to go deep formechanism studies

Conflict of Interests

The authors report no conflict of interests

Acknowledgments

The Authors would like to thank the Department of Micro-biology Gulbarga University Gulbarga India for provid-ing logistic support and facilities They also thank SAIF-STIC Cochin Ruska labs Hyderabad SAIF-IIT Madras andMalvern Instruments Ltd Bangalore India for characteri-zation studies

References

[1] CMarambio-Jones and EM VHoek ldquoA review of the antibac-terial effects of silver nanomaterials and potential implicationsfor human health and the environmentrdquo Journal of NanoparticleResearch vol 12 no 5 pp 1531ndash1551 2010

[2] D Rejeski ldquoNanotechnology and consumer productsrdquohttpwwwnanotechprojectorgpublicationsarchivenano-technology consumer products

[3] D Singh V Rathod N Shivaraj J Hiremath A K Singhand J Methew ldquoOptimization and characterization of silvernanoparticles by endophytic fungi Penicillium sp isolated from

Curcuma longa (turmeric) and Application studies againstMDR E coli and S aureusrdquo Bioinorganic Chemistry and Appli-cations vol 2014 Article ID 408021 8 pages 2014

[4] A Banu V Rathod and E Ranganath ldquoSilver nanoparticleproduction by Rhizopus stolonifer and its antibacterial activ-ity against extended spectrum 120573-lactamase producing (ESBL)strains of Enterobacteriaceaerdquo Materials Research Bulletin vol46 no 9 pp 1417ndash1423 2011

[5] D Singh V Rathod N Shivaraj J Hiremath and P Kulka-rni ldquoBiosynthesis of silver nanoparticles by endophytic fungiPenicillium sp isolated from Curcuma longa (turmeric) and itsantibacterial activity against pathogenic gram negative bacte-riardquo Journal of Pharmacy Research vol 7 no 5 pp 448ndash4532013

[6] V Rathod A Banu and E Ranganath ldquoBiosynthesis of highlystabilized silver nanoparticles by Rhizopus stolonifer and theirAnti-fungal efficacyrdquo International Journal of Molecular andClinical Microbiology vol 1 no 2 pp 65ndash70 2011

[7] S Galdiero A Falanga M Vitiello M Cantisani V Marra andM Galdiero ldquoSilver nanoparticles as potential antiviral agentsrdquoMolecules vol 16 no 10 pp 8894ndash8918 2011

[8] Y Wang L Cao S Guan et al ldquoSilver mineralization onself-assembled peptide nanofibers for long term antimicrobialeffectrdquo Journal of Materials Chemistry vol 22 no 6 pp 2575ndash2581 2012

[9] W-R Li X-B Xie Q-S Shi H-Y Zeng Y-S Ou-Yang andY-B Chen ldquoAntibacterial activity and mechanism of silvernanoparticles on Escherichia colirdquo Applied Microbiology andBiotechnology vol 85 no 4 pp 1115ndash1122 2010

[10] J M Galloway and S S Staniland ldquoProtein and peptidebiotemplated metal and metal oxide nanoparticles and theirpatterning onto surfacesrdquo Journal of Materials Chemistry vol22 no 25 pp 12423ndash12434 2012

[11] P Mohanpuria N K Rana and S K Yadav ldquoBiosynthesis ofnanoparticles technological concepts and future applicationsrdquoJournal of Nanoparticle Research vol 10 no 3 pp 507ndash517 2008

[12] S K Das and E Marsili ldquoA green chemical approach for thesynthesis of gold nanoparticles characterization andmechanis-tic aspectrdquo Reviews in Environmental Science and Biotechnologyvol 9 no 3 pp 199ndash204 2010

[13] I Sondi and B Salopek-Sondi ldquoSilver nanoparticles as antimi-crobial agent a case study on E coli as a model for Gram-negative bacteriardquo Journal of Colloid and Interface Science vol275 no 1 pp 177ndash182 2004

[14] S-H Kim H-S Lee D-S Ryu S-J Choi and D-S LeeldquoAntibacterial activity of silver-nanoparticles against Staphylo-coccus aureus and Escherichia colirdquo Korean Journal of Microbiol-ogy and Biotechnology vol 39 no 1 pp 77ndash85 2011

BioMed Research International 9

[15] A J Kora and J Arunachalam ldquoAssessment of antibacterialactivity of silver nanoparticles on Pseudomonas aeruginosa andits mechanism of actionrdquo World Journal of Microbiology andBiotechnology vol 27 no 5 pp 1209ndash1216 2011

[16] J S Kim E Kuk K N Yu et al ldquoAntimicrobial effects of silvernanoparticlesrdquo Nanomedicine vol 3 no 1 pp 95ndash101 2007

[17] U K Parashar V Kumar T Bera et al ldquoStudy of mechanismof enhanced antibacterial activity by green synthesis of silvernanoparticlesrdquo Nanotechnology vol 22 no 41 Article ID415104 13 pages 2011

[18] S Bonde ldquoA Biogenic approach for green synthesis of silvernanoparticles using extract of Foeniculum vulgare and itsactivity against S aureus and Ecolirdquo Bioscience vol 3 no 1 pp59ndash63 2011

[19] J Hiremath V Rathod S Ninganagouda D Singh and KPrema ldquoAntibacterial activity of Silver Nanoparticles from Rhi-zopus spp against Gram negative EcoliMDR strainsrdquo Journal ofPure and Applied Microbiology vol 8 no 1 pp 555ndash562 2014

[20] Y Zhou Y Kong S Kundu J D Cirillo and H LiangldquoAntibacterial activities of gold and silver nanoparticles againstEscherichia coli and bacillus Calmette-Guerinrdquo Journal ofNanobiotechnology vol 10 article 19 2012

[21] W K Jung H C Koo K W Kim S Shin S H Kim and YH Park ldquoAntibacterial activity and mechanism of action of thesilver ion in Staphylococcus aureus and Escherichia colirdquoAppliedand Environmental Microbiology vol 74 no 7 pp 2171ndash21782008

[22] M Yamanaka K Hara and J Kudo ldquoBactericidal actions ofa silver ion solution on Escherichia coli studied by energy-filtering transmission electron microscopy and proteomic anal-ysisrdquoApplied and EnvironmentalMicrobiology vol 71 no 11 pp7589ndash7593 2005

[23] Q L Feng J Wu G Q Chen T N Kim and J O Kim ldquoAmechanistic study of the antibacterial effect of silver ions onEscherichia coli and Staphylococcus aureusrdquo Journal of Biomedi-cal Materials Research vol 52 no 4 pp 662ndash668 2000

[24] J R Morones J L Elechiguerra A Camacho et al ldquoThebactericidal effect of silver nanoparticlesrdquo Nanotechnology vol16 no 10 pp 2346ndash2353 2005

[25] Y Matsumura K Yoshikata S-I Kunisaki and T TsuchidoldquoMode of bactericidal action of silver zeolite and its comparisonwith that of silver nitraterdquo Applied and Environmental Microbi-ology vol 69 no 7 pp 4278ndash4281 2003

[26] J A Lemire J JHarrison andR J Turner ldquoAntimicrobial activ-ity of metals mechanisms molecular targets and applicationsrdquoNature Reviews Microbiology vol 11 no 6 pp 371ndash384 2013

[27] AGrigorEva I Saranina N Tikunova et al ldquoFinemechanismsof the interaction of silver nanoparticles with the cells ofSalmonella typhimurium and Staphylococcus aureusrdquo BioMetalsvol 26 no 3 pp 479ndash488 2013

[28] M Chen Z Yang H Wu X Pan X Xie and C WuldquoAntimicrobial activity and the mechanism of silver nanoparti-cle thermosensitive gelrdquo International Journal of Nanomedicinevol 6 pp 2873ndash2877 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 3: Research Article Growth Kinetics and Mechanistic Action of ...downloads.hindawi.com/journals/bmri/2014/753419.pdf · Species Released by Silver Nanoparticles from Aspergillus niger

BioMed Research International 3

1080

0893

0707

0521

0335

0148

3000 3800 4600 5400 6200 7000

Abso

rban

ce (a

bs)

WL (nm)

12

(3795 0837)

(4165 0885)1

2(3795 0837)

(4165 0885)

Figure 1 UV-Vis-spectrum of silver nanoparticles

52855

53241

53608

55931

77875

108010116044

122736131348

138645

153860

161381

177102216123

292801

329073

525660

6468

7276808488

92

5001000150020002500300035004000

T(

)

Wavenumbers (cmminus1)

Figure 2 FTIR spectra of silver nanoparticles from A niger

450 cmminus1 revealed the presence of different functional groupslike 329073mdashsecondary amide (NndashH stretch H-bonded)292801mdashalkane (CndashH stretching) 216123mdashalkyne (CequivCstretching) 177102mdashanhydride (C=O stretching) 161381mdashalkene (C=C stretching) 153860mdasharomatic (CndashC stretch-ing) 138645 131348mdash and 108010mdashprimary alcohol (CndashO stretching) and 52855mdashalkene (=CndashH bending) respec-tively Rathod et al [6] reported that proteins present inthe extract of fungus Rhizopus stolonifer can bind to theAgNPs through either free amino or carboxyl groups in theproteins and also they reported different functional groupsabsorbing characteristic frequencies of FTIR radiation Andalso our result correlates with Parashar et al [17] whoreported the possible reaction mechanism for the reductionof Ag+ usingGuara (Psidium guajara) leaf extractThus FTIRis an important and popular tool for structural elucidationand compound identification

A drop of AgNPs solution was placed on the carboncoated copper grids and kept under vacuum before loadingthem onto a specimen holder Then TEM micrographs weretaken by prepared grids to determine the size and shape of theproducedAgNPs Figure 3 revealed the particles are sphericalin shape and size of the particles is between 20 and 55 nmRathod et al [6] synthesized AgNPs from Rhizopus stolonifer

Figure 3 TEMmicrograph of silver nanoparticles synthesized usingextracellular filtrate of A niger

and reported that the size is ranging between 5 and 50 nmsuggesting that biological molecules could possibly performthe function for the stabilization of the AgNPs and alsoreported that the AgNPs synthesized by this route are fairlystable even after prolonged storage Size and shape of AgNPsas revealed by Kora and Arunachalam [15] were 45 nm

The particle size distribution of the AgNPs was shownunder different categories like size distribution by volumeand by intensity The average diameter of the particleswas found to be 73 nm (100 intensity) (Figure 4(a)) witha zeta potential that was minus24mV (Figure 4(b)) The syn-thesized AgNps are well distributed with respect to vol-ume and intensity indicates the well dispersion of AgNPsThe average diameter of the particles was found to be127 100 intensity and width was found to be 3725 nmshowing monodispersity of the particles using the extractof the plant Foeniculum vulgare as reported by Bonde[18]

Scanning electron microscopy (SEM) was used to recordthe photomicrograph images of synthesized AgNPs A smallvolume of AgNPs suspension was taken for SEM analysis onelectromicroscope stub The stubs were placed briefly in adrier and then coated with gold in an ion sputter Pictureswere taken by random scanning of the stub Shape and surfacemorphology of AgNPs were studied by SEM Figure 5(a)shows the distribution of AgNPs SEM micrograph of singlenanoparticle reveals the spherical and smoothmorphology ofthe nanoparticle (Figure 5(b))

Energy dispersive spectroscopy (EDS) samples were pre-pared on a copper substrate by drop coating of AgNPs Theelemental analysis was examined by EDS The presence ofan optical absorption band at sim3 eV reveals the presenceof pure metallic AgNPs along with the C and O signaturesthat might be from the stabilizing protein (Figure 5) Asour result correlates with Li et al [9] who reported theoptical absorption peak at 3 kev from commercially availableAgNPs EDS analysis gives the additional evidence for thereduction of AgNPs the optical absorption peak at 3 kevreported by Parashar et al [17] is typical for the absorptionof metallic AgNPs due to surface Plasmon resonance whichconfirms the presence of nanocrystalline elemental silver (seeFigure 6)

4 BioMed Research International

12

10

8

6

4

2

001 1 10 100 1000 10000

Size distribution by intensity

Inte

nsity

()

Record 10 sample-02 1

Size (dmiddotnm)

(a)

200000

150000

100000

50000

0

minus100 0 100 200

Zeta potential distribution

Apparent zeta potential (mV)

Tota

l cou

nts

Record 10 sample-02 1

(b)

Figure 4 (a) Particle size distribution of AgNPs by intensity with Zeta analyzer (b) Zeta potential

(a) (b)

Figure 5 (a) SEM micrograph of silver nanoparticles (b) single nanoparticle with spherical and smooth morphology

CKa

OKa

NaK

a

AuM

zAu

Ma

AuM

r

AgL

aA

gLb AgL

b2 CaKa

CaKb

AuL1 Au

La

1000

900

800

700

600

500

400

300

200

0

000 100 200 300 400 500 600 700 800 900 1000

Cou

nts

100

C1Kb

C1Ka

(keV)

Figure 6 EDS spectrumof extracellular synthesized silver nanopar-ticles

32 Bactericidal Activities of AgNPs The bactericidal activitywas performed against Gram negative E coli on NA platescontaining different concentrations of AgNPs such as 0 255 10 and 15 120583gmL (Figure 7) Number of bacterial coloniesgrew on NA plate as a function of concentration of AgNPs

when overnight E coli culture was applied to the plates Thepresence of AgNPs at concentrations of 10 and 15120583gmLinhibited bacterial growth by 100 There was luxuriousgrowth at 0120583gmL that is without AgNPs while at 25 120583gmLof AgNPs there was substantial growth maybe because theconcentration of 25120583gmL of AgNPs is not sufficient to killE coli while at 5 120583gmL there was decreased growth dueto enhanced AgNPs concentration Our results indicate that10 120583gmL of AgNPs are sufficient to inhibit 107 CFUmL of Ecoli For confirmation we went for 15 120583gmL concentration ofAgNPs againstE coliHere also therewas complete inhibitionthat indicates that a concentration of 10120583gmL of AgNPs issufficient to completely kill 107 CFUmL of E coli

Li et al [9] also reported that 10 120583gmL of AgNPs aresufficient to completely inhibit 107 CFUmL of E coli thoughthe AgNPs were not biologically synthesized but purchasedcommercially yet our results correlate with them Sondiand Salopek-Sondi [13] also reported that a concentrationof 20120583gmL cmminus3 completely prevented bacterial growth if104 CFU of E coli were used While Kim et al [14] reporteda MIC of 100 120583gmL of AgNPs to cause complete death of Saureus and E coli Comparing our results with the above saidby authors AgNPs produced by A niger are more effectivein completely inhibiting E coli at the minimum dose of

BioMed Research International 5

5120583gmL 15120583gmL

0120583gmL

25 120583gmL

10120583gmL

Figure 7 Bactericidal activities of AgNPs on E coli at different concentrations ranging from 0 120583gmL to 15120583gmL

25

2

15

1

05

0

minus05

5 10 15 20 25

Time (h)

0120583gmL

5120583gmL25 120583gmL

10120583gmL15120583gmL

Abso

rban

ce

Figure 8 Growth curve of E coli treated with AgNPs at 0 120583gmLmaximum growth at 25 120583gmL growth reduced at 5120583gmL bac-terial growth still reduced and at 10 and 15 120583gmL growth curvesdiminished showing nil growth

10 120583gmL Hiremath et al [19] also reported antibacterialactivity ofAgNPs againstMDRE coli strains using the fungusRhizopus sp

33 Growth Curves of Bacterial Cells Treated with AgNPs Thegrowth curve of E coli treated with AgNPs was determinedby using NB supplemented with 0 25 5 10 and 15120583gmLof AgNPs The concentrations of 25 and 5 120583gmL showedthe growth of E coli but comparatively less than the growthcurve shown without AgNPs (0120583gmL) It is very interestingto observe that the incorporations of 10 and 15 120583gmL AgNPsto NB showed complete inhibition of E coli which is evidentfrom graph in Figure 8 The dynamics of bacterial growthwas monitored in liquid LB broth supplemented with 107 Ecoli cells with 10 50 and 100 120583g cmminus3 of AgNPs at all theseconcentrations caused a growth delay of E coli Zhou et al[20] also reported that a concentration of 10 120583gmL of AgNPsshowed strong antibacterial activity against E coli

34 Release of ROS from the Surface of AgNPs and ItsMechanism Gram negative E coli was selected as a model

6 BioMed Research International

250nm

(a)

200nm

(b)

200nm

(c)

200nm

(d)

150nm

(e)

200nm

(f)

Figure 9 TEMmicrograph of E coli loaded with AgNPs (a) Normal E coli cell with its well-integrated cell wall (b) Anchoring of AgNPs oncell wall of E coli (c) Ruptured cell membrane and entry of AgNPs into the cytoplasm (d) Complete damage of cell wall and cell membrane(e) Clear AgNPs within the E coli cell (f) Complete destruction of E coli cell and overloading of AgNPs

to study the effect of ROS released from the surface of AgNPson the permeability and the membrane structure of E colicells The interaction of AgNPs with bacterium was analyzedusing TEMmicrographs E coli samples were incubated withAgNPs for 12 hours and were analyzed by employing TEMFigure 9(a) shows normal E coli cells with its well-integratedcell wall When the E coli cells were made to interact withAgNPs for 1 hr it can be seen fromFigure 9(b) that theAgNPswere trying to adhere to the surface of E coli cells After 5 hrsof interaction a closure look at the bacterial cell membranereveals that AgNPs anchored onto the cell surface of E coliandmany pits and gaps appeared in themicrograph and theirmembrane was fragmented (Figure 9(c)) Figure 9(d) shows

that AgNPs are trying to get inside the bacterial cell throughthe ruptured cell membrane After 8 hours the AgNPssurround the bacterial cell surface and almost cover the sidesof the cell and nanoparticles are visible in the cytoplasm alsoIn addition electron dense particles or precipitates were alsoobserved around the damaged bacterial cell (Figure 9(e))The interaction can either completely disintegrates the cell ormay cause cell lyses after 12 hours Figure 9(f) shows AgNPseventually leading to the bacterial cell death

Another proposed mechanism of E colimembrane dam-age byAgNPs relates tometal depletion that is the formationof pits in the outer membrane and change in membranepermeability by the progressive release of lipopolysaccharide

BioMed Research International 7

2047

minus2048

327640 (mT)

(A) 337475 g = 200025

337640 (mT) 347640 (mT)

(m1) 332715 g = 202887(m2 minus m1) = 87037 (mT)

(m2) 341418 g = 197715

Figure 10 ESR spectrum of AgNPs recorded at room temperaturem1

(332715) and m2

(341418) indicates the control peak of Mnand the peak (mT 337475) indicates the released free radical fromAgNPs Instrument setting JEOL-JES-TE 200 spectrophotometermicropower 4mW MOD 100 khz and the time const 003 sec

20E+06

15E+06

10E+06

50E+05

0

500 600 700 800

Wavelength (nm)

Emission acquistion

Inte

nsity

(cps

)

File 1 = VRSRJ01

Figure 11 Formation of ROS in E coli using fluorescence spec-troscopy

(LPS) molecules and membrane proteins A bacterial mem-brane with this morphology exhibits a significant increase inpermeability leaving the bacterial cells incapable of properlyregulating transport through the plasma membrane andfinally causing cell death It is well known that the outermembrane of E coli cells is predominantly constructed fromtightly packed LPS molecules which provide an effectivepermeability barrier [13] It has been also proposed that thesites of interaction for AgNPs and membrane cells might bedue to sulfur containing proteins in a similar way as silverinteracts with thiol groups of respiratory chain proteins andtransport proteins interfering with their proper function [21ndash25]

Reactive Oxygen Species (ROS) are natural byproducts ofthemetabolismof respiring organisms [16] Induction of ROSsynthesis leads to the formation of highly reactive radicalsthat destroy the cells The bacterial growth inhibition byformation of free radicals from the surface of AgNPs was alsoobserved by employing ESR spectroscopy (Figure 10) Excessgeneration of reactive oxygen species can attack membranelipids and then lead to a breakdown of membrane functionCertain transition metals might disrupt the cellular donorligands that coordinate Fe Mounting evidence suggests that

the primary targets for variousmetals are the solvent exposed[4Fe-4S] clusters of proteins The direct or indirect destruc-tion of [4Fe-4S] clusters by metals could result in the releaseof additional Fenton-active Fe into the cytoplasm resultingin increased ROS formation The ability to induce Fe releasefrom these proteins as well as from other Fe-containingproteins might account for observations that some Fenton-inactive metals (such as Ag Hg and Ga) generate ROS andthat cells require or upregulate ROS-detoxification enzymesto withstand toxic doses of these nanoparticles [26]The ROSproduction from the surface of AgNPs was measured usingDCFH-DAmethod Dichlorofluorescein diacetate (DCFDA)is a popular fluorescence-based probe for reactive oxygenspecies (ROS) detection in vitro After 5 h of incubationROS formed in the sample was detected at 523 nm of emis-sion wavelength using Fluorescence spectroscopy (Figure 11)Intracellular esterases cleave DCFH-DA at the two esterbonds producing a relatively polar and cell membrane-impermeable product H2DCFThis nonfluorescentmoleculeaccumulates intracellularly and subsequent oxidation yieldsthe highly fluorescent product DCF The redox state of thesample can bemonitored by detecting the increase in fluores-cence The result confirms the generation of free radicals andfrom the surface of AgNPs which becomes toxic to bacterialcells leading to death Our result corroborates with Kim etal [14] who reported the oxidative stress can cause damageto bacterial cell membrane protein structure and intracel-luluar system against S aureus and E coli usingDCFDAmethod

To determine the involvement of ROS in the antibacterialactivity of AgNPs we used ascorbic acid as scavenger Thisantioxidant was used to scavenge the ROS produced bythe AgNPs Protective activity of antioxidant against bac-tericidal activity of AgNPs was observed in Figure 12 Incontrol plate the bacterial colonies were clearly seen withoutantioxidants and AgNPs but in the plate supplementedwith AgNPs (10 120583gmL) no bacterial growth was observedrevealing that AgNPs completely inhibited bacterial growthdue to ROS formation Surprisingly the bacterial colonieswere observed in the plate supplemented with both AgNPsand antioxidant which clearly indicates that the ascorbicacid used as an antioxidant serves as a scavenger hinderingthe ROS release from AgNPs It is determined that theantioxidant prevents the formation of a silver oxide layeron the AgNPs surface and consequently formation of theAg+ reservoir Kora and Arunachalam [15] reported the100 P aeruginosa growth survival by using ascorbic acidas scavenger while 73 of bacteria protected from NAC asscavenger

Bacterial cells exposed to AgNPs suffer morphologicalchanges such as cytoplasm shrinkage detachment of cellwall membrane DNA condensation and localization in anelectron-light region in the centre of the cell and cellmembrane degradation allowing the leakage of intracellularcontents [14 27] Physiological changes occur together withthe morphological changes bacterial cells enter an activebut nonculturable state in which physiological levels can bemeasured but cells are not able to grow and replicate [9 28]

8 BioMed Research International

Control SNPs SNPs + ascorbic acid

Figure 12 Antioxidant activities of silver nanoparticles on E coli

4 Conclusion

From our results two aspects can be elucidated one is thefact that AgNPs themselves having antibacterial propertiesact on the pathogenic bacteria by anchoring to the cell surfacetrying to get inside the bacterial cell through ruptured cellmembrane leading to the perforation of cell membrane bycausing leakage of cell components and finally cell deathThesecond aspect is the release of ROS from AgNPs producedby A niger which is clearly evident from the facts thatthe antioxidant ascorbic acid is used as a scavenger ROSproductionwas confirmed by usingDCFH-DAmethodusingfluorescence spectroscopy Antioxidant will inhibit ROS pro-duction thereby luxurious growth of E coli was observedNutrient agar plate inoculated with E coli supplemented withAgNPs without addition of antioxidant showed completeinhibition of E coli suggesting that ROS is released fromAgNPs which completely inhibit E coli Our studies suggestthat 10 120583gmL of AgNPs are sufficient for complete inhibitionof E coli which is a boon to biotechnologists to go deep formechanism studies

Conflict of Interests

The authors report no conflict of interests

Acknowledgments

The Authors would like to thank the Department of Micro-biology Gulbarga University Gulbarga India for provid-ing logistic support and facilities They also thank SAIF-STIC Cochin Ruska labs Hyderabad SAIF-IIT Madras andMalvern Instruments Ltd Bangalore India for characteri-zation studies

References

[1] CMarambio-Jones and EM VHoek ldquoA review of the antibac-terial effects of silver nanomaterials and potential implicationsfor human health and the environmentrdquo Journal of NanoparticleResearch vol 12 no 5 pp 1531ndash1551 2010

[2] D Rejeski ldquoNanotechnology and consumer productsrdquohttpwwwnanotechprojectorgpublicationsarchivenano-technology consumer products

[3] D Singh V Rathod N Shivaraj J Hiremath A K Singhand J Methew ldquoOptimization and characterization of silvernanoparticles by endophytic fungi Penicillium sp isolated from

Curcuma longa (turmeric) and Application studies againstMDR E coli and S aureusrdquo Bioinorganic Chemistry and Appli-cations vol 2014 Article ID 408021 8 pages 2014

[4] A Banu V Rathod and E Ranganath ldquoSilver nanoparticleproduction by Rhizopus stolonifer and its antibacterial activ-ity against extended spectrum 120573-lactamase producing (ESBL)strains of Enterobacteriaceaerdquo Materials Research Bulletin vol46 no 9 pp 1417ndash1423 2011

[5] D Singh V Rathod N Shivaraj J Hiremath and P Kulka-rni ldquoBiosynthesis of silver nanoparticles by endophytic fungiPenicillium sp isolated from Curcuma longa (turmeric) and itsantibacterial activity against pathogenic gram negative bacte-riardquo Journal of Pharmacy Research vol 7 no 5 pp 448ndash4532013

[6] V Rathod A Banu and E Ranganath ldquoBiosynthesis of highlystabilized silver nanoparticles by Rhizopus stolonifer and theirAnti-fungal efficacyrdquo International Journal of Molecular andClinical Microbiology vol 1 no 2 pp 65ndash70 2011

[7] S Galdiero A Falanga M Vitiello M Cantisani V Marra andM Galdiero ldquoSilver nanoparticles as potential antiviral agentsrdquoMolecules vol 16 no 10 pp 8894ndash8918 2011

[8] Y Wang L Cao S Guan et al ldquoSilver mineralization onself-assembled peptide nanofibers for long term antimicrobialeffectrdquo Journal of Materials Chemistry vol 22 no 6 pp 2575ndash2581 2012

[9] W-R Li X-B Xie Q-S Shi H-Y Zeng Y-S Ou-Yang andY-B Chen ldquoAntibacterial activity and mechanism of silvernanoparticles on Escherichia colirdquo Applied Microbiology andBiotechnology vol 85 no 4 pp 1115ndash1122 2010

[10] J M Galloway and S S Staniland ldquoProtein and peptidebiotemplated metal and metal oxide nanoparticles and theirpatterning onto surfacesrdquo Journal of Materials Chemistry vol22 no 25 pp 12423ndash12434 2012

[11] P Mohanpuria N K Rana and S K Yadav ldquoBiosynthesis ofnanoparticles technological concepts and future applicationsrdquoJournal of Nanoparticle Research vol 10 no 3 pp 507ndash517 2008

[12] S K Das and E Marsili ldquoA green chemical approach for thesynthesis of gold nanoparticles characterization andmechanis-tic aspectrdquo Reviews in Environmental Science and Biotechnologyvol 9 no 3 pp 199ndash204 2010

[13] I Sondi and B Salopek-Sondi ldquoSilver nanoparticles as antimi-crobial agent a case study on E coli as a model for Gram-negative bacteriardquo Journal of Colloid and Interface Science vol275 no 1 pp 177ndash182 2004

[14] S-H Kim H-S Lee D-S Ryu S-J Choi and D-S LeeldquoAntibacterial activity of silver-nanoparticles against Staphylo-coccus aureus and Escherichia colirdquo Korean Journal of Microbiol-ogy and Biotechnology vol 39 no 1 pp 77ndash85 2011

BioMed Research International 9

[15] A J Kora and J Arunachalam ldquoAssessment of antibacterialactivity of silver nanoparticles on Pseudomonas aeruginosa andits mechanism of actionrdquo World Journal of Microbiology andBiotechnology vol 27 no 5 pp 1209ndash1216 2011

[16] J S Kim E Kuk K N Yu et al ldquoAntimicrobial effects of silvernanoparticlesrdquo Nanomedicine vol 3 no 1 pp 95ndash101 2007

[17] U K Parashar V Kumar T Bera et al ldquoStudy of mechanismof enhanced antibacterial activity by green synthesis of silvernanoparticlesrdquo Nanotechnology vol 22 no 41 Article ID415104 13 pages 2011

[18] S Bonde ldquoA Biogenic approach for green synthesis of silvernanoparticles using extract of Foeniculum vulgare and itsactivity against S aureus and Ecolirdquo Bioscience vol 3 no 1 pp59ndash63 2011

[19] J Hiremath V Rathod S Ninganagouda D Singh and KPrema ldquoAntibacterial activity of Silver Nanoparticles from Rhi-zopus spp against Gram negative EcoliMDR strainsrdquo Journal ofPure and Applied Microbiology vol 8 no 1 pp 555ndash562 2014

[20] Y Zhou Y Kong S Kundu J D Cirillo and H LiangldquoAntibacterial activities of gold and silver nanoparticles againstEscherichia coli and bacillus Calmette-Guerinrdquo Journal ofNanobiotechnology vol 10 article 19 2012

[21] W K Jung H C Koo K W Kim S Shin S H Kim and YH Park ldquoAntibacterial activity and mechanism of action of thesilver ion in Staphylococcus aureus and Escherichia colirdquoAppliedand Environmental Microbiology vol 74 no 7 pp 2171ndash21782008

[22] M Yamanaka K Hara and J Kudo ldquoBactericidal actions ofa silver ion solution on Escherichia coli studied by energy-filtering transmission electron microscopy and proteomic anal-ysisrdquoApplied and EnvironmentalMicrobiology vol 71 no 11 pp7589ndash7593 2005

[23] Q L Feng J Wu G Q Chen T N Kim and J O Kim ldquoAmechanistic study of the antibacterial effect of silver ions onEscherichia coli and Staphylococcus aureusrdquo Journal of Biomedi-cal Materials Research vol 52 no 4 pp 662ndash668 2000

[24] J R Morones J L Elechiguerra A Camacho et al ldquoThebactericidal effect of silver nanoparticlesrdquo Nanotechnology vol16 no 10 pp 2346ndash2353 2005

[25] Y Matsumura K Yoshikata S-I Kunisaki and T TsuchidoldquoMode of bactericidal action of silver zeolite and its comparisonwith that of silver nitraterdquo Applied and Environmental Microbi-ology vol 69 no 7 pp 4278ndash4281 2003

[26] J A Lemire J JHarrison andR J Turner ldquoAntimicrobial activ-ity of metals mechanisms molecular targets and applicationsrdquoNature Reviews Microbiology vol 11 no 6 pp 371ndash384 2013

[27] AGrigorEva I Saranina N Tikunova et al ldquoFinemechanismsof the interaction of silver nanoparticles with the cells ofSalmonella typhimurium and Staphylococcus aureusrdquo BioMetalsvol 26 no 3 pp 479ndash488 2013

[28] M Chen Z Yang H Wu X Pan X Xie and C WuldquoAntimicrobial activity and the mechanism of silver nanoparti-cle thermosensitive gelrdquo International Journal of Nanomedicinevol 6 pp 2873ndash2877 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 4: Research Article Growth Kinetics and Mechanistic Action of ...downloads.hindawi.com/journals/bmri/2014/753419.pdf · Species Released by Silver Nanoparticles from Aspergillus niger

4 BioMed Research International

12

10

8

6

4

2

001 1 10 100 1000 10000

Size distribution by intensity

Inte

nsity

()

Record 10 sample-02 1

Size (dmiddotnm)

(a)

200000

150000

100000

50000

0

minus100 0 100 200

Zeta potential distribution

Apparent zeta potential (mV)

Tota

l cou

nts

Record 10 sample-02 1

(b)

Figure 4 (a) Particle size distribution of AgNPs by intensity with Zeta analyzer (b) Zeta potential

(a) (b)

Figure 5 (a) SEM micrograph of silver nanoparticles (b) single nanoparticle with spherical and smooth morphology

CKa

OKa

NaK

a

AuM

zAu

Ma

AuM

r

AgL

aA

gLb AgL

b2 CaKa

CaKb

AuL1 Au

La

1000

900

800

700

600

500

400

300

200

0

000 100 200 300 400 500 600 700 800 900 1000

Cou

nts

100

C1Kb

C1Ka

(keV)

Figure 6 EDS spectrumof extracellular synthesized silver nanopar-ticles

32 Bactericidal Activities of AgNPs The bactericidal activitywas performed against Gram negative E coli on NA platescontaining different concentrations of AgNPs such as 0 255 10 and 15 120583gmL (Figure 7) Number of bacterial coloniesgrew on NA plate as a function of concentration of AgNPs

when overnight E coli culture was applied to the plates Thepresence of AgNPs at concentrations of 10 and 15120583gmLinhibited bacterial growth by 100 There was luxuriousgrowth at 0120583gmL that is without AgNPs while at 25 120583gmLof AgNPs there was substantial growth maybe because theconcentration of 25120583gmL of AgNPs is not sufficient to killE coli while at 5 120583gmL there was decreased growth dueto enhanced AgNPs concentration Our results indicate that10 120583gmL of AgNPs are sufficient to inhibit 107 CFUmL of Ecoli For confirmation we went for 15 120583gmL concentration ofAgNPs againstE coliHere also therewas complete inhibitionthat indicates that a concentration of 10120583gmL of AgNPs issufficient to completely kill 107 CFUmL of E coli

Li et al [9] also reported that 10 120583gmL of AgNPs aresufficient to completely inhibit 107 CFUmL of E coli thoughthe AgNPs were not biologically synthesized but purchasedcommercially yet our results correlate with them Sondiand Salopek-Sondi [13] also reported that a concentrationof 20120583gmL cmminus3 completely prevented bacterial growth if104 CFU of E coli were used While Kim et al [14] reporteda MIC of 100 120583gmL of AgNPs to cause complete death of Saureus and E coli Comparing our results with the above saidby authors AgNPs produced by A niger are more effectivein completely inhibiting E coli at the minimum dose of

BioMed Research International 5

5120583gmL 15120583gmL

0120583gmL

25 120583gmL

10120583gmL

Figure 7 Bactericidal activities of AgNPs on E coli at different concentrations ranging from 0 120583gmL to 15120583gmL

25

2

15

1

05

0

minus05

5 10 15 20 25

Time (h)

0120583gmL

5120583gmL25 120583gmL

10120583gmL15120583gmL

Abso

rban

ce

Figure 8 Growth curve of E coli treated with AgNPs at 0 120583gmLmaximum growth at 25 120583gmL growth reduced at 5120583gmL bac-terial growth still reduced and at 10 and 15 120583gmL growth curvesdiminished showing nil growth

10 120583gmL Hiremath et al [19] also reported antibacterialactivity ofAgNPs againstMDRE coli strains using the fungusRhizopus sp

33 Growth Curves of Bacterial Cells Treated with AgNPs Thegrowth curve of E coli treated with AgNPs was determinedby using NB supplemented with 0 25 5 10 and 15120583gmLof AgNPs The concentrations of 25 and 5 120583gmL showedthe growth of E coli but comparatively less than the growthcurve shown without AgNPs (0120583gmL) It is very interestingto observe that the incorporations of 10 and 15 120583gmL AgNPsto NB showed complete inhibition of E coli which is evidentfrom graph in Figure 8 The dynamics of bacterial growthwas monitored in liquid LB broth supplemented with 107 Ecoli cells with 10 50 and 100 120583g cmminus3 of AgNPs at all theseconcentrations caused a growth delay of E coli Zhou et al[20] also reported that a concentration of 10 120583gmL of AgNPsshowed strong antibacterial activity against E coli

34 Release of ROS from the Surface of AgNPs and ItsMechanism Gram negative E coli was selected as a model

6 BioMed Research International

250nm

(a)

200nm

(b)

200nm

(c)

200nm

(d)

150nm

(e)

200nm

(f)

Figure 9 TEMmicrograph of E coli loaded with AgNPs (a) Normal E coli cell with its well-integrated cell wall (b) Anchoring of AgNPs oncell wall of E coli (c) Ruptured cell membrane and entry of AgNPs into the cytoplasm (d) Complete damage of cell wall and cell membrane(e) Clear AgNPs within the E coli cell (f) Complete destruction of E coli cell and overloading of AgNPs

to study the effect of ROS released from the surface of AgNPson the permeability and the membrane structure of E colicells The interaction of AgNPs with bacterium was analyzedusing TEMmicrographs E coli samples were incubated withAgNPs for 12 hours and were analyzed by employing TEMFigure 9(a) shows normal E coli cells with its well-integratedcell wall When the E coli cells were made to interact withAgNPs for 1 hr it can be seen fromFigure 9(b) that theAgNPswere trying to adhere to the surface of E coli cells After 5 hrsof interaction a closure look at the bacterial cell membranereveals that AgNPs anchored onto the cell surface of E coliandmany pits and gaps appeared in themicrograph and theirmembrane was fragmented (Figure 9(c)) Figure 9(d) shows

that AgNPs are trying to get inside the bacterial cell throughthe ruptured cell membrane After 8 hours the AgNPssurround the bacterial cell surface and almost cover the sidesof the cell and nanoparticles are visible in the cytoplasm alsoIn addition electron dense particles or precipitates were alsoobserved around the damaged bacterial cell (Figure 9(e))The interaction can either completely disintegrates the cell ormay cause cell lyses after 12 hours Figure 9(f) shows AgNPseventually leading to the bacterial cell death

Another proposed mechanism of E colimembrane dam-age byAgNPs relates tometal depletion that is the formationof pits in the outer membrane and change in membranepermeability by the progressive release of lipopolysaccharide

BioMed Research International 7

2047

minus2048

327640 (mT)

(A) 337475 g = 200025

337640 (mT) 347640 (mT)

(m1) 332715 g = 202887(m2 minus m1) = 87037 (mT)

(m2) 341418 g = 197715

Figure 10 ESR spectrum of AgNPs recorded at room temperaturem1

(332715) and m2

(341418) indicates the control peak of Mnand the peak (mT 337475) indicates the released free radical fromAgNPs Instrument setting JEOL-JES-TE 200 spectrophotometermicropower 4mW MOD 100 khz and the time const 003 sec

20E+06

15E+06

10E+06

50E+05

0

500 600 700 800

Wavelength (nm)

Emission acquistion

Inte

nsity

(cps

)

File 1 = VRSRJ01

Figure 11 Formation of ROS in E coli using fluorescence spec-troscopy

(LPS) molecules and membrane proteins A bacterial mem-brane with this morphology exhibits a significant increase inpermeability leaving the bacterial cells incapable of properlyregulating transport through the plasma membrane andfinally causing cell death It is well known that the outermembrane of E coli cells is predominantly constructed fromtightly packed LPS molecules which provide an effectivepermeability barrier [13] It has been also proposed that thesites of interaction for AgNPs and membrane cells might bedue to sulfur containing proteins in a similar way as silverinteracts with thiol groups of respiratory chain proteins andtransport proteins interfering with their proper function [21ndash25]

Reactive Oxygen Species (ROS) are natural byproducts ofthemetabolismof respiring organisms [16] Induction of ROSsynthesis leads to the formation of highly reactive radicalsthat destroy the cells The bacterial growth inhibition byformation of free radicals from the surface of AgNPs was alsoobserved by employing ESR spectroscopy (Figure 10) Excessgeneration of reactive oxygen species can attack membranelipids and then lead to a breakdown of membrane functionCertain transition metals might disrupt the cellular donorligands that coordinate Fe Mounting evidence suggests that

the primary targets for variousmetals are the solvent exposed[4Fe-4S] clusters of proteins The direct or indirect destruc-tion of [4Fe-4S] clusters by metals could result in the releaseof additional Fenton-active Fe into the cytoplasm resultingin increased ROS formation The ability to induce Fe releasefrom these proteins as well as from other Fe-containingproteins might account for observations that some Fenton-inactive metals (such as Ag Hg and Ga) generate ROS andthat cells require or upregulate ROS-detoxification enzymesto withstand toxic doses of these nanoparticles [26]The ROSproduction from the surface of AgNPs was measured usingDCFH-DAmethod Dichlorofluorescein diacetate (DCFDA)is a popular fluorescence-based probe for reactive oxygenspecies (ROS) detection in vitro After 5 h of incubationROS formed in the sample was detected at 523 nm of emis-sion wavelength using Fluorescence spectroscopy (Figure 11)Intracellular esterases cleave DCFH-DA at the two esterbonds producing a relatively polar and cell membrane-impermeable product H2DCFThis nonfluorescentmoleculeaccumulates intracellularly and subsequent oxidation yieldsthe highly fluorescent product DCF The redox state of thesample can bemonitored by detecting the increase in fluores-cence The result confirms the generation of free radicals andfrom the surface of AgNPs which becomes toxic to bacterialcells leading to death Our result corroborates with Kim etal [14] who reported the oxidative stress can cause damageto bacterial cell membrane protein structure and intracel-luluar system against S aureus and E coli usingDCFDAmethod

To determine the involvement of ROS in the antibacterialactivity of AgNPs we used ascorbic acid as scavenger Thisantioxidant was used to scavenge the ROS produced bythe AgNPs Protective activity of antioxidant against bac-tericidal activity of AgNPs was observed in Figure 12 Incontrol plate the bacterial colonies were clearly seen withoutantioxidants and AgNPs but in the plate supplementedwith AgNPs (10 120583gmL) no bacterial growth was observedrevealing that AgNPs completely inhibited bacterial growthdue to ROS formation Surprisingly the bacterial colonieswere observed in the plate supplemented with both AgNPsand antioxidant which clearly indicates that the ascorbicacid used as an antioxidant serves as a scavenger hinderingthe ROS release from AgNPs It is determined that theantioxidant prevents the formation of a silver oxide layeron the AgNPs surface and consequently formation of theAg+ reservoir Kora and Arunachalam [15] reported the100 P aeruginosa growth survival by using ascorbic acidas scavenger while 73 of bacteria protected from NAC asscavenger

Bacterial cells exposed to AgNPs suffer morphologicalchanges such as cytoplasm shrinkage detachment of cellwall membrane DNA condensation and localization in anelectron-light region in the centre of the cell and cellmembrane degradation allowing the leakage of intracellularcontents [14 27] Physiological changes occur together withthe morphological changes bacterial cells enter an activebut nonculturable state in which physiological levels can bemeasured but cells are not able to grow and replicate [9 28]

8 BioMed Research International

Control SNPs SNPs + ascorbic acid

Figure 12 Antioxidant activities of silver nanoparticles on E coli

4 Conclusion

From our results two aspects can be elucidated one is thefact that AgNPs themselves having antibacterial propertiesact on the pathogenic bacteria by anchoring to the cell surfacetrying to get inside the bacterial cell through ruptured cellmembrane leading to the perforation of cell membrane bycausing leakage of cell components and finally cell deathThesecond aspect is the release of ROS from AgNPs producedby A niger which is clearly evident from the facts thatthe antioxidant ascorbic acid is used as a scavenger ROSproductionwas confirmed by usingDCFH-DAmethodusingfluorescence spectroscopy Antioxidant will inhibit ROS pro-duction thereby luxurious growth of E coli was observedNutrient agar plate inoculated with E coli supplemented withAgNPs without addition of antioxidant showed completeinhibition of E coli suggesting that ROS is released fromAgNPs which completely inhibit E coli Our studies suggestthat 10 120583gmL of AgNPs are sufficient for complete inhibitionof E coli which is a boon to biotechnologists to go deep formechanism studies

Conflict of Interests

The authors report no conflict of interests

Acknowledgments

The Authors would like to thank the Department of Micro-biology Gulbarga University Gulbarga India for provid-ing logistic support and facilities They also thank SAIF-STIC Cochin Ruska labs Hyderabad SAIF-IIT Madras andMalvern Instruments Ltd Bangalore India for characteri-zation studies

References

[1] CMarambio-Jones and EM VHoek ldquoA review of the antibac-terial effects of silver nanomaterials and potential implicationsfor human health and the environmentrdquo Journal of NanoparticleResearch vol 12 no 5 pp 1531ndash1551 2010

[2] D Rejeski ldquoNanotechnology and consumer productsrdquohttpwwwnanotechprojectorgpublicationsarchivenano-technology consumer products

[3] D Singh V Rathod N Shivaraj J Hiremath A K Singhand J Methew ldquoOptimization and characterization of silvernanoparticles by endophytic fungi Penicillium sp isolated from

Curcuma longa (turmeric) and Application studies againstMDR E coli and S aureusrdquo Bioinorganic Chemistry and Appli-cations vol 2014 Article ID 408021 8 pages 2014

[4] A Banu V Rathod and E Ranganath ldquoSilver nanoparticleproduction by Rhizopus stolonifer and its antibacterial activ-ity against extended spectrum 120573-lactamase producing (ESBL)strains of Enterobacteriaceaerdquo Materials Research Bulletin vol46 no 9 pp 1417ndash1423 2011

[5] D Singh V Rathod N Shivaraj J Hiremath and P Kulka-rni ldquoBiosynthesis of silver nanoparticles by endophytic fungiPenicillium sp isolated from Curcuma longa (turmeric) and itsantibacterial activity against pathogenic gram negative bacte-riardquo Journal of Pharmacy Research vol 7 no 5 pp 448ndash4532013

[6] V Rathod A Banu and E Ranganath ldquoBiosynthesis of highlystabilized silver nanoparticles by Rhizopus stolonifer and theirAnti-fungal efficacyrdquo International Journal of Molecular andClinical Microbiology vol 1 no 2 pp 65ndash70 2011

[7] S Galdiero A Falanga M Vitiello M Cantisani V Marra andM Galdiero ldquoSilver nanoparticles as potential antiviral agentsrdquoMolecules vol 16 no 10 pp 8894ndash8918 2011

[8] Y Wang L Cao S Guan et al ldquoSilver mineralization onself-assembled peptide nanofibers for long term antimicrobialeffectrdquo Journal of Materials Chemistry vol 22 no 6 pp 2575ndash2581 2012

[9] W-R Li X-B Xie Q-S Shi H-Y Zeng Y-S Ou-Yang andY-B Chen ldquoAntibacterial activity and mechanism of silvernanoparticles on Escherichia colirdquo Applied Microbiology andBiotechnology vol 85 no 4 pp 1115ndash1122 2010

[10] J M Galloway and S S Staniland ldquoProtein and peptidebiotemplated metal and metal oxide nanoparticles and theirpatterning onto surfacesrdquo Journal of Materials Chemistry vol22 no 25 pp 12423ndash12434 2012

[11] P Mohanpuria N K Rana and S K Yadav ldquoBiosynthesis ofnanoparticles technological concepts and future applicationsrdquoJournal of Nanoparticle Research vol 10 no 3 pp 507ndash517 2008

[12] S K Das and E Marsili ldquoA green chemical approach for thesynthesis of gold nanoparticles characterization andmechanis-tic aspectrdquo Reviews in Environmental Science and Biotechnologyvol 9 no 3 pp 199ndash204 2010

[13] I Sondi and B Salopek-Sondi ldquoSilver nanoparticles as antimi-crobial agent a case study on E coli as a model for Gram-negative bacteriardquo Journal of Colloid and Interface Science vol275 no 1 pp 177ndash182 2004

[14] S-H Kim H-S Lee D-S Ryu S-J Choi and D-S LeeldquoAntibacterial activity of silver-nanoparticles against Staphylo-coccus aureus and Escherichia colirdquo Korean Journal of Microbiol-ogy and Biotechnology vol 39 no 1 pp 77ndash85 2011

BioMed Research International 9

[15] A J Kora and J Arunachalam ldquoAssessment of antibacterialactivity of silver nanoparticles on Pseudomonas aeruginosa andits mechanism of actionrdquo World Journal of Microbiology andBiotechnology vol 27 no 5 pp 1209ndash1216 2011

[16] J S Kim E Kuk K N Yu et al ldquoAntimicrobial effects of silvernanoparticlesrdquo Nanomedicine vol 3 no 1 pp 95ndash101 2007

[17] U K Parashar V Kumar T Bera et al ldquoStudy of mechanismof enhanced antibacterial activity by green synthesis of silvernanoparticlesrdquo Nanotechnology vol 22 no 41 Article ID415104 13 pages 2011

[18] S Bonde ldquoA Biogenic approach for green synthesis of silvernanoparticles using extract of Foeniculum vulgare and itsactivity against S aureus and Ecolirdquo Bioscience vol 3 no 1 pp59ndash63 2011

[19] J Hiremath V Rathod S Ninganagouda D Singh and KPrema ldquoAntibacterial activity of Silver Nanoparticles from Rhi-zopus spp against Gram negative EcoliMDR strainsrdquo Journal ofPure and Applied Microbiology vol 8 no 1 pp 555ndash562 2014

[20] Y Zhou Y Kong S Kundu J D Cirillo and H LiangldquoAntibacterial activities of gold and silver nanoparticles againstEscherichia coli and bacillus Calmette-Guerinrdquo Journal ofNanobiotechnology vol 10 article 19 2012

[21] W K Jung H C Koo K W Kim S Shin S H Kim and YH Park ldquoAntibacterial activity and mechanism of action of thesilver ion in Staphylococcus aureus and Escherichia colirdquoAppliedand Environmental Microbiology vol 74 no 7 pp 2171ndash21782008

[22] M Yamanaka K Hara and J Kudo ldquoBactericidal actions ofa silver ion solution on Escherichia coli studied by energy-filtering transmission electron microscopy and proteomic anal-ysisrdquoApplied and EnvironmentalMicrobiology vol 71 no 11 pp7589ndash7593 2005

[23] Q L Feng J Wu G Q Chen T N Kim and J O Kim ldquoAmechanistic study of the antibacterial effect of silver ions onEscherichia coli and Staphylococcus aureusrdquo Journal of Biomedi-cal Materials Research vol 52 no 4 pp 662ndash668 2000

[24] J R Morones J L Elechiguerra A Camacho et al ldquoThebactericidal effect of silver nanoparticlesrdquo Nanotechnology vol16 no 10 pp 2346ndash2353 2005

[25] Y Matsumura K Yoshikata S-I Kunisaki and T TsuchidoldquoMode of bactericidal action of silver zeolite and its comparisonwith that of silver nitraterdquo Applied and Environmental Microbi-ology vol 69 no 7 pp 4278ndash4281 2003

[26] J A Lemire J JHarrison andR J Turner ldquoAntimicrobial activ-ity of metals mechanisms molecular targets and applicationsrdquoNature Reviews Microbiology vol 11 no 6 pp 371ndash384 2013

[27] AGrigorEva I Saranina N Tikunova et al ldquoFinemechanismsof the interaction of silver nanoparticles with the cells ofSalmonella typhimurium and Staphylococcus aureusrdquo BioMetalsvol 26 no 3 pp 479ndash488 2013

[28] M Chen Z Yang H Wu X Pan X Xie and C WuldquoAntimicrobial activity and the mechanism of silver nanoparti-cle thermosensitive gelrdquo International Journal of Nanomedicinevol 6 pp 2873ndash2877 2011

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

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StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

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Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

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Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 5: Research Article Growth Kinetics and Mechanistic Action of ...downloads.hindawi.com/journals/bmri/2014/753419.pdf · Species Released by Silver Nanoparticles from Aspergillus niger

BioMed Research International 5

5120583gmL 15120583gmL

0120583gmL

25 120583gmL

10120583gmL

Figure 7 Bactericidal activities of AgNPs on E coli at different concentrations ranging from 0 120583gmL to 15120583gmL

25

2

15

1

05

0

minus05

5 10 15 20 25

Time (h)

0120583gmL

5120583gmL25 120583gmL

10120583gmL15120583gmL

Abso

rban

ce

Figure 8 Growth curve of E coli treated with AgNPs at 0 120583gmLmaximum growth at 25 120583gmL growth reduced at 5120583gmL bac-terial growth still reduced and at 10 and 15 120583gmL growth curvesdiminished showing nil growth

10 120583gmL Hiremath et al [19] also reported antibacterialactivity ofAgNPs againstMDRE coli strains using the fungusRhizopus sp

33 Growth Curves of Bacterial Cells Treated with AgNPs Thegrowth curve of E coli treated with AgNPs was determinedby using NB supplemented with 0 25 5 10 and 15120583gmLof AgNPs The concentrations of 25 and 5 120583gmL showedthe growth of E coli but comparatively less than the growthcurve shown without AgNPs (0120583gmL) It is very interestingto observe that the incorporations of 10 and 15 120583gmL AgNPsto NB showed complete inhibition of E coli which is evidentfrom graph in Figure 8 The dynamics of bacterial growthwas monitored in liquid LB broth supplemented with 107 Ecoli cells with 10 50 and 100 120583g cmminus3 of AgNPs at all theseconcentrations caused a growth delay of E coli Zhou et al[20] also reported that a concentration of 10 120583gmL of AgNPsshowed strong antibacterial activity against E coli

34 Release of ROS from the Surface of AgNPs and ItsMechanism Gram negative E coli was selected as a model

6 BioMed Research International

250nm

(a)

200nm

(b)

200nm

(c)

200nm

(d)

150nm

(e)

200nm

(f)

Figure 9 TEMmicrograph of E coli loaded with AgNPs (a) Normal E coli cell with its well-integrated cell wall (b) Anchoring of AgNPs oncell wall of E coli (c) Ruptured cell membrane and entry of AgNPs into the cytoplasm (d) Complete damage of cell wall and cell membrane(e) Clear AgNPs within the E coli cell (f) Complete destruction of E coli cell and overloading of AgNPs

to study the effect of ROS released from the surface of AgNPson the permeability and the membrane structure of E colicells The interaction of AgNPs with bacterium was analyzedusing TEMmicrographs E coli samples were incubated withAgNPs for 12 hours and were analyzed by employing TEMFigure 9(a) shows normal E coli cells with its well-integratedcell wall When the E coli cells were made to interact withAgNPs for 1 hr it can be seen fromFigure 9(b) that theAgNPswere trying to adhere to the surface of E coli cells After 5 hrsof interaction a closure look at the bacterial cell membranereveals that AgNPs anchored onto the cell surface of E coliandmany pits and gaps appeared in themicrograph and theirmembrane was fragmented (Figure 9(c)) Figure 9(d) shows

that AgNPs are trying to get inside the bacterial cell throughthe ruptured cell membrane After 8 hours the AgNPssurround the bacterial cell surface and almost cover the sidesof the cell and nanoparticles are visible in the cytoplasm alsoIn addition electron dense particles or precipitates were alsoobserved around the damaged bacterial cell (Figure 9(e))The interaction can either completely disintegrates the cell ormay cause cell lyses after 12 hours Figure 9(f) shows AgNPseventually leading to the bacterial cell death

Another proposed mechanism of E colimembrane dam-age byAgNPs relates tometal depletion that is the formationof pits in the outer membrane and change in membranepermeability by the progressive release of lipopolysaccharide

BioMed Research International 7

2047

minus2048

327640 (mT)

(A) 337475 g = 200025

337640 (mT) 347640 (mT)

(m1) 332715 g = 202887(m2 minus m1) = 87037 (mT)

(m2) 341418 g = 197715

Figure 10 ESR spectrum of AgNPs recorded at room temperaturem1

(332715) and m2

(341418) indicates the control peak of Mnand the peak (mT 337475) indicates the released free radical fromAgNPs Instrument setting JEOL-JES-TE 200 spectrophotometermicropower 4mW MOD 100 khz and the time const 003 sec

20E+06

15E+06

10E+06

50E+05

0

500 600 700 800

Wavelength (nm)

Emission acquistion

Inte

nsity

(cps

)

File 1 = VRSRJ01

Figure 11 Formation of ROS in E coli using fluorescence spec-troscopy

(LPS) molecules and membrane proteins A bacterial mem-brane with this morphology exhibits a significant increase inpermeability leaving the bacterial cells incapable of properlyregulating transport through the plasma membrane andfinally causing cell death It is well known that the outermembrane of E coli cells is predominantly constructed fromtightly packed LPS molecules which provide an effectivepermeability barrier [13] It has been also proposed that thesites of interaction for AgNPs and membrane cells might bedue to sulfur containing proteins in a similar way as silverinteracts with thiol groups of respiratory chain proteins andtransport proteins interfering with their proper function [21ndash25]

Reactive Oxygen Species (ROS) are natural byproducts ofthemetabolismof respiring organisms [16] Induction of ROSsynthesis leads to the formation of highly reactive radicalsthat destroy the cells The bacterial growth inhibition byformation of free radicals from the surface of AgNPs was alsoobserved by employing ESR spectroscopy (Figure 10) Excessgeneration of reactive oxygen species can attack membranelipids and then lead to a breakdown of membrane functionCertain transition metals might disrupt the cellular donorligands that coordinate Fe Mounting evidence suggests that

the primary targets for variousmetals are the solvent exposed[4Fe-4S] clusters of proteins The direct or indirect destruc-tion of [4Fe-4S] clusters by metals could result in the releaseof additional Fenton-active Fe into the cytoplasm resultingin increased ROS formation The ability to induce Fe releasefrom these proteins as well as from other Fe-containingproteins might account for observations that some Fenton-inactive metals (such as Ag Hg and Ga) generate ROS andthat cells require or upregulate ROS-detoxification enzymesto withstand toxic doses of these nanoparticles [26]The ROSproduction from the surface of AgNPs was measured usingDCFH-DAmethod Dichlorofluorescein diacetate (DCFDA)is a popular fluorescence-based probe for reactive oxygenspecies (ROS) detection in vitro After 5 h of incubationROS formed in the sample was detected at 523 nm of emis-sion wavelength using Fluorescence spectroscopy (Figure 11)Intracellular esterases cleave DCFH-DA at the two esterbonds producing a relatively polar and cell membrane-impermeable product H2DCFThis nonfluorescentmoleculeaccumulates intracellularly and subsequent oxidation yieldsthe highly fluorescent product DCF The redox state of thesample can bemonitored by detecting the increase in fluores-cence The result confirms the generation of free radicals andfrom the surface of AgNPs which becomes toxic to bacterialcells leading to death Our result corroborates with Kim etal [14] who reported the oxidative stress can cause damageto bacterial cell membrane protein structure and intracel-luluar system against S aureus and E coli usingDCFDAmethod

To determine the involvement of ROS in the antibacterialactivity of AgNPs we used ascorbic acid as scavenger Thisantioxidant was used to scavenge the ROS produced bythe AgNPs Protective activity of antioxidant against bac-tericidal activity of AgNPs was observed in Figure 12 Incontrol plate the bacterial colonies were clearly seen withoutantioxidants and AgNPs but in the plate supplementedwith AgNPs (10 120583gmL) no bacterial growth was observedrevealing that AgNPs completely inhibited bacterial growthdue to ROS formation Surprisingly the bacterial colonieswere observed in the plate supplemented with both AgNPsand antioxidant which clearly indicates that the ascorbicacid used as an antioxidant serves as a scavenger hinderingthe ROS release from AgNPs It is determined that theantioxidant prevents the formation of a silver oxide layeron the AgNPs surface and consequently formation of theAg+ reservoir Kora and Arunachalam [15] reported the100 P aeruginosa growth survival by using ascorbic acidas scavenger while 73 of bacteria protected from NAC asscavenger

Bacterial cells exposed to AgNPs suffer morphologicalchanges such as cytoplasm shrinkage detachment of cellwall membrane DNA condensation and localization in anelectron-light region in the centre of the cell and cellmembrane degradation allowing the leakage of intracellularcontents [14 27] Physiological changes occur together withthe morphological changes bacterial cells enter an activebut nonculturable state in which physiological levels can bemeasured but cells are not able to grow and replicate [9 28]

8 BioMed Research International

Control SNPs SNPs + ascorbic acid

Figure 12 Antioxidant activities of silver nanoparticles on E coli

4 Conclusion

From our results two aspects can be elucidated one is thefact that AgNPs themselves having antibacterial propertiesact on the pathogenic bacteria by anchoring to the cell surfacetrying to get inside the bacterial cell through ruptured cellmembrane leading to the perforation of cell membrane bycausing leakage of cell components and finally cell deathThesecond aspect is the release of ROS from AgNPs producedby A niger which is clearly evident from the facts thatthe antioxidant ascorbic acid is used as a scavenger ROSproductionwas confirmed by usingDCFH-DAmethodusingfluorescence spectroscopy Antioxidant will inhibit ROS pro-duction thereby luxurious growth of E coli was observedNutrient agar plate inoculated with E coli supplemented withAgNPs without addition of antioxidant showed completeinhibition of E coli suggesting that ROS is released fromAgNPs which completely inhibit E coli Our studies suggestthat 10 120583gmL of AgNPs are sufficient for complete inhibitionof E coli which is a boon to biotechnologists to go deep formechanism studies

Conflict of Interests

The authors report no conflict of interests

Acknowledgments

The Authors would like to thank the Department of Micro-biology Gulbarga University Gulbarga India for provid-ing logistic support and facilities They also thank SAIF-STIC Cochin Ruska labs Hyderabad SAIF-IIT Madras andMalvern Instruments Ltd Bangalore India for characteri-zation studies

References

[1] CMarambio-Jones and EM VHoek ldquoA review of the antibac-terial effects of silver nanomaterials and potential implicationsfor human health and the environmentrdquo Journal of NanoparticleResearch vol 12 no 5 pp 1531ndash1551 2010

[2] D Rejeski ldquoNanotechnology and consumer productsrdquohttpwwwnanotechprojectorgpublicationsarchivenano-technology consumer products

[3] D Singh V Rathod N Shivaraj J Hiremath A K Singhand J Methew ldquoOptimization and characterization of silvernanoparticles by endophytic fungi Penicillium sp isolated from

Curcuma longa (turmeric) and Application studies againstMDR E coli and S aureusrdquo Bioinorganic Chemistry and Appli-cations vol 2014 Article ID 408021 8 pages 2014

[4] A Banu V Rathod and E Ranganath ldquoSilver nanoparticleproduction by Rhizopus stolonifer and its antibacterial activ-ity against extended spectrum 120573-lactamase producing (ESBL)strains of Enterobacteriaceaerdquo Materials Research Bulletin vol46 no 9 pp 1417ndash1423 2011

[5] D Singh V Rathod N Shivaraj J Hiremath and P Kulka-rni ldquoBiosynthesis of silver nanoparticles by endophytic fungiPenicillium sp isolated from Curcuma longa (turmeric) and itsantibacterial activity against pathogenic gram negative bacte-riardquo Journal of Pharmacy Research vol 7 no 5 pp 448ndash4532013

[6] V Rathod A Banu and E Ranganath ldquoBiosynthesis of highlystabilized silver nanoparticles by Rhizopus stolonifer and theirAnti-fungal efficacyrdquo International Journal of Molecular andClinical Microbiology vol 1 no 2 pp 65ndash70 2011

[7] S Galdiero A Falanga M Vitiello M Cantisani V Marra andM Galdiero ldquoSilver nanoparticles as potential antiviral agentsrdquoMolecules vol 16 no 10 pp 8894ndash8918 2011

[8] Y Wang L Cao S Guan et al ldquoSilver mineralization onself-assembled peptide nanofibers for long term antimicrobialeffectrdquo Journal of Materials Chemistry vol 22 no 6 pp 2575ndash2581 2012

[9] W-R Li X-B Xie Q-S Shi H-Y Zeng Y-S Ou-Yang andY-B Chen ldquoAntibacterial activity and mechanism of silvernanoparticles on Escherichia colirdquo Applied Microbiology andBiotechnology vol 85 no 4 pp 1115ndash1122 2010

[10] J M Galloway and S S Staniland ldquoProtein and peptidebiotemplated metal and metal oxide nanoparticles and theirpatterning onto surfacesrdquo Journal of Materials Chemistry vol22 no 25 pp 12423ndash12434 2012

[11] P Mohanpuria N K Rana and S K Yadav ldquoBiosynthesis ofnanoparticles technological concepts and future applicationsrdquoJournal of Nanoparticle Research vol 10 no 3 pp 507ndash517 2008

[12] S K Das and E Marsili ldquoA green chemical approach for thesynthesis of gold nanoparticles characterization andmechanis-tic aspectrdquo Reviews in Environmental Science and Biotechnologyvol 9 no 3 pp 199ndash204 2010

[13] I Sondi and B Salopek-Sondi ldquoSilver nanoparticles as antimi-crobial agent a case study on E coli as a model for Gram-negative bacteriardquo Journal of Colloid and Interface Science vol275 no 1 pp 177ndash182 2004

[14] S-H Kim H-S Lee D-S Ryu S-J Choi and D-S LeeldquoAntibacterial activity of silver-nanoparticles against Staphylo-coccus aureus and Escherichia colirdquo Korean Journal of Microbiol-ogy and Biotechnology vol 39 no 1 pp 77ndash85 2011

BioMed Research International 9

[15] A J Kora and J Arunachalam ldquoAssessment of antibacterialactivity of silver nanoparticles on Pseudomonas aeruginosa andits mechanism of actionrdquo World Journal of Microbiology andBiotechnology vol 27 no 5 pp 1209ndash1216 2011

[16] J S Kim E Kuk K N Yu et al ldquoAntimicrobial effects of silvernanoparticlesrdquo Nanomedicine vol 3 no 1 pp 95ndash101 2007

[17] U K Parashar V Kumar T Bera et al ldquoStudy of mechanismof enhanced antibacterial activity by green synthesis of silvernanoparticlesrdquo Nanotechnology vol 22 no 41 Article ID415104 13 pages 2011

[18] S Bonde ldquoA Biogenic approach for green synthesis of silvernanoparticles using extract of Foeniculum vulgare and itsactivity against S aureus and Ecolirdquo Bioscience vol 3 no 1 pp59ndash63 2011

[19] J Hiremath V Rathod S Ninganagouda D Singh and KPrema ldquoAntibacterial activity of Silver Nanoparticles from Rhi-zopus spp against Gram negative EcoliMDR strainsrdquo Journal ofPure and Applied Microbiology vol 8 no 1 pp 555ndash562 2014

[20] Y Zhou Y Kong S Kundu J D Cirillo and H LiangldquoAntibacterial activities of gold and silver nanoparticles againstEscherichia coli and bacillus Calmette-Guerinrdquo Journal ofNanobiotechnology vol 10 article 19 2012

[21] W K Jung H C Koo K W Kim S Shin S H Kim and YH Park ldquoAntibacterial activity and mechanism of action of thesilver ion in Staphylococcus aureus and Escherichia colirdquoAppliedand Environmental Microbiology vol 74 no 7 pp 2171ndash21782008

[22] M Yamanaka K Hara and J Kudo ldquoBactericidal actions ofa silver ion solution on Escherichia coli studied by energy-filtering transmission electron microscopy and proteomic anal-ysisrdquoApplied and EnvironmentalMicrobiology vol 71 no 11 pp7589ndash7593 2005

[23] Q L Feng J Wu G Q Chen T N Kim and J O Kim ldquoAmechanistic study of the antibacterial effect of silver ions onEscherichia coli and Staphylococcus aureusrdquo Journal of Biomedi-cal Materials Research vol 52 no 4 pp 662ndash668 2000

[24] J R Morones J L Elechiguerra A Camacho et al ldquoThebactericidal effect of silver nanoparticlesrdquo Nanotechnology vol16 no 10 pp 2346ndash2353 2005

[25] Y Matsumura K Yoshikata S-I Kunisaki and T TsuchidoldquoMode of bactericidal action of silver zeolite and its comparisonwith that of silver nitraterdquo Applied and Environmental Microbi-ology vol 69 no 7 pp 4278ndash4281 2003

[26] J A Lemire J JHarrison andR J Turner ldquoAntimicrobial activ-ity of metals mechanisms molecular targets and applicationsrdquoNature Reviews Microbiology vol 11 no 6 pp 371ndash384 2013

[27] AGrigorEva I Saranina N Tikunova et al ldquoFinemechanismsof the interaction of silver nanoparticles with the cells ofSalmonella typhimurium and Staphylococcus aureusrdquo BioMetalsvol 26 no 3 pp 479ndash488 2013

[28] M Chen Z Yang H Wu X Pan X Xie and C WuldquoAntimicrobial activity and the mechanism of silver nanoparti-cle thermosensitive gelrdquo International Journal of Nanomedicinevol 6 pp 2873ndash2877 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 6: Research Article Growth Kinetics and Mechanistic Action of ...downloads.hindawi.com/journals/bmri/2014/753419.pdf · Species Released by Silver Nanoparticles from Aspergillus niger

6 BioMed Research International

250nm

(a)

200nm

(b)

200nm

(c)

200nm

(d)

150nm

(e)

200nm

(f)

Figure 9 TEMmicrograph of E coli loaded with AgNPs (a) Normal E coli cell with its well-integrated cell wall (b) Anchoring of AgNPs oncell wall of E coli (c) Ruptured cell membrane and entry of AgNPs into the cytoplasm (d) Complete damage of cell wall and cell membrane(e) Clear AgNPs within the E coli cell (f) Complete destruction of E coli cell and overloading of AgNPs

to study the effect of ROS released from the surface of AgNPson the permeability and the membrane structure of E colicells The interaction of AgNPs with bacterium was analyzedusing TEMmicrographs E coli samples were incubated withAgNPs for 12 hours and were analyzed by employing TEMFigure 9(a) shows normal E coli cells with its well-integratedcell wall When the E coli cells were made to interact withAgNPs for 1 hr it can be seen fromFigure 9(b) that theAgNPswere trying to adhere to the surface of E coli cells After 5 hrsof interaction a closure look at the bacterial cell membranereveals that AgNPs anchored onto the cell surface of E coliandmany pits and gaps appeared in themicrograph and theirmembrane was fragmented (Figure 9(c)) Figure 9(d) shows

that AgNPs are trying to get inside the bacterial cell throughthe ruptured cell membrane After 8 hours the AgNPssurround the bacterial cell surface and almost cover the sidesof the cell and nanoparticles are visible in the cytoplasm alsoIn addition electron dense particles or precipitates were alsoobserved around the damaged bacterial cell (Figure 9(e))The interaction can either completely disintegrates the cell ormay cause cell lyses after 12 hours Figure 9(f) shows AgNPseventually leading to the bacterial cell death

Another proposed mechanism of E colimembrane dam-age byAgNPs relates tometal depletion that is the formationof pits in the outer membrane and change in membranepermeability by the progressive release of lipopolysaccharide

BioMed Research International 7

2047

minus2048

327640 (mT)

(A) 337475 g = 200025

337640 (mT) 347640 (mT)

(m1) 332715 g = 202887(m2 minus m1) = 87037 (mT)

(m2) 341418 g = 197715

Figure 10 ESR spectrum of AgNPs recorded at room temperaturem1

(332715) and m2

(341418) indicates the control peak of Mnand the peak (mT 337475) indicates the released free radical fromAgNPs Instrument setting JEOL-JES-TE 200 spectrophotometermicropower 4mW MOD 100 khz and the time const 003 sec

20E+06

15E+06

10E+06

50E+05

0

500 600 700 800

Wavelength (nm)

Emission acquistion

Inte

nsity

(cps

)

File 1 = VRSRJ01

Figure 11 Formation of ROS in E coli using fluorescence spec-troscopy

(LPS) molecules and membrane proteins A bacterial mem-brane with this morphology exhibits a significant increase inpermeability leaving the bacterial cells incapable of properlyregulating transport through the plasma membrane andfinally causing cell death It is well known that the outermembrane of E coli cells is predominantly constructed fromtightly packed LPS molecules which provide an effectivepermeability barrier [13] It has been also proposed that thesites of interaction for AgNPs and membrane cells might bedue to sulfur containing proteins in a similar way as silverinteracts with thiol groups of respiratory chain proteins andtransport proteins interfering with their proper function [21ndash25]

Reactive Oxygen Species (ROS) are natural byproducts ofthemetabolismof respiring organisms [16] Induction of ROSsynthesis leads to the formation of highly reactive radicalsthat destroy the cells The bacterial growth inhibition byformation of free radicals from the surface of AgNPs was alsoobserved by employing ESR spectroscopy (Figure 10) Excessgeneration of reactive oxygen species can attack membranelipids and then lead to a breakdown of membrane functionCertain transition metals might disrupt the cellular donorligands that coordinate Fe Mounting evidence suggests that

the primary targets for variousmetals are the solvent exposed[4Fe-4S] clusters of proteins The direct or indirect destruc-tion of [4Fe-4S] clusters by metals could result in the releaseof additional Fenton-active Fe into the cytoplasm resultingin increased ROS formation The ability to induce Fe releasefrom these proteins as well as from other Fe-containingproteins might account for observations that some Fenton-inactive metals (such as Ag Hg and Ga) generate ROS andthat cells require or upregulate ROS-detoxification enzymesto withstand toxic doses of these nanoparticles [26]The ROSproduction from the surface of AgNPs was measured usingDCFH-DAmethod Dichlorofluorescein diacetate (DCFDA)is a popular fluorescence-based probe for reactive oxygenspecies (ROS) detection in vitro After 5 h of incubationROS formed in the sample was detected at 523 nm of emis-sion wavelength using Fluorescence spectroscopy (Figure 11)Intracellular esterases cleave DCFH-DA at the two esterbonds producing a relatively polar and cell membrane-impermeable product H2DCFThis nonfluorescentmoleculeaccumulates intracellularly and subsequent oxidation yieldsthe highly fluorescent product DCF The redox state of thesample can bemonitored by detecting the increase in fluores-cence The result confirms the generation of free radicals andfrom the surface of AgNPs which becomes toxic to bacterialcells leading to death Our result corroborates with Kim etal [14] who reported the oxidative stress can cause damageto bacterial cell membrane protein structure and intracel-luluar system against S aureus and E coli usingDCFDAmethod

To determine the involvement of ROS in the antibacterialactivity of AgNPs we used ascorbic acid as scavenger Thisantioxidant was used to scavenge the ROS produced bythe AgNPs Protective activity of antioxidant against bac-tericidal activity of AgNPs was observed in Figure 12 Incontrol plate the bacterial colonies were clearly seen withoutantioxidants and AgNPs but in the plate supplementedwith AgNPs (10 120583gmL) no bacterial growth was observedrevealing that AgNPs completely inhibited bacterial growthdue to ROS formation Surprisingly the bacterial colonieswere observed in the plate supplemented with both AgNPsand antioxidant which clearly indicates that the ascorbicacid used as an antioxidant serves as a scavenger hinderingthe ROS release from AgNPs It is determined that theantioxidant prevents the formation of a silver oxide layeron the AgNPs surface and consequently formation of theAg+ reservoir Kora and Arunachalam [15] reported the100 P aeruginosa growth survival by using ascorbic acidas scavenger while 73 of bacteria protected from NAC asscavenger

Bacterial cells exposed to AgNPs suffer morphologicalchanges such as cytoplasm shrinkage detachment of cellwall membrane DNA condensation and localization in anelectron-light region in the centre of the cell and cellmembrane degradation allowing the leakage of intracellularcontents [14 27] Physiological changes occur together withthe morphological changes bacterial cells enter an activebut nonculturable state in which physiological levels can bemeasured but cells are not able to grow and replicate [9 28]

8 BioMed Research International

Control SNPs SNPs + ascorbic acid

Figure 12 Antioxidant activities of silver nanoparticles on E coli

4 Conclusion

From our results two aspects can be elucidated one is thefact that AgNPs themselves having antibacterial propertiesact on the pathogenic bacteria by anchoring to the cell surfacetrying to get inside the bacterial cell through ruptured cellmembrane leading to the perforation of cell membrane bycausing leakage of cell components and finally cell deathThesecond aspect is the release of ROS from AgNPs producedby A niger which is clearly evident from the facts thatthe antioxidant ascorbic acid is used as a scavenger ROSproductionwas confirmed by usingDCFH-DAmethodusingfluorescence spectroscopy Antioxidant will inhibit ROS pro-duction thereby luxurious growth of E coli was observedNutrient agar plate inoculated with E coli supplemented withAgNPs without addition of antioxidant showed completeinhibition of E coli suggesting that ROS is released fromAgNPs which completely inhibit E coli Our studies suggestthat 10 120583gmL of AgNPs are sufficient for complete inhibitionof E coli which is a boon to biotechnologists to go deep formechanism studies

Conflict of Interests

The authors report no conflict of interests

Acknowledgments

The Authors would like to thank the Department of Micro-biology Gulbarga University Gulbarga India for provid-ing logistic support and facilities They also thank SAIF-STIC Cochin Ruska labs Hyderabad SAIF-IIT Madras andMalvern Instruments Ltd Bangalore India for characteri-zation studies

References

[1] CMarambio-Jones and EM VHoek ldquoA review of the antibac-terial effects of silver nanomaterials and potential implicationsfor human health and the environmentrdquo Journal of NanoparticleResearch vol 12 no 5 pp 1531ndash1551 2010

[2] D Rejeski ldquoNanotechnology and consumer productsrdquohttpwwwnanotechprojectorgpublicationsarchivenano-technology consumer products

[3] D Singh V Rathod N Shivaraj J Hiremath A K Singhand J Methew ldquoOptimization and characterization of silvernanoparticles by endophytic fungi Penicillium sp isolated from

Curcuma longa (turmeric) and Application studies againstMDR E coli and S aureusrdquo Bioinorganic Chemistry and Appli-cations vol 2014 Article ID 408021 8 pages 2014

[4] A Banu V Rathod and E Ranganath ldquoSilver nanoparticleproduction by Rhizopus stolonifer and its antibacterial activ-ity against extended spectrum 120573-lactamase producing (ESBL)strains of Enterobacteriaceaerdquo Materials Research Bulletin vol46 no 9 pp 1417ndash1423 2011

[5] D Singh V Rathod N Shivaraj J Hiremath and P Kulka-rni ldquoBiosynthesis of silver nanoparticles by endophytic fungiPenicillium sp isolated from Curcuma longa (turmeric) and itsantibacterial activity against pathogenic gram negative bacte-riardquo Journal of Pharmacy Research vol 7 no 5 pp 448ndash4532013

[6] V Rathod A Banu and E Ranganath ldquoBiosynthesis of highlystabilized silver nanoparticles by Rhizopus stolonifer and theirAnti-fungal efficacyrdquo International Journal of Molecular andClinical Microbiology vol 1 no 2 pp 65ndash70 2011

[7] S Galdiero A Falanga M Vitiello M Cantisani V Marra andM Galdiero ldquoSilver nanoparticles as potential antiviral agentsrdquoMolecules vol 16 no 10 pp 8894ndash8918 2011

[8] Y Wang L Cao S Guan et al ldquoSilver mineralization onself-assembled peptide nanofibers for long term antimicrobialeffectrdquo Journal of Materials Chemistry vol 22 no 6 pp 2575ndash2581 2012

[9] W-R Li X-B Xie Q-S Shi H-Y Zeng Y-S Ou-Yang andY-B Chen ldquoAntibacterial activity and mechanism of silvernanoparticles on Escherichia colirdquo Applied Microbiology andBiotechnology vol 85 no 4 pp 1115ndash1122 2010

[10] J M Galloway and S S Staniland ldquoProtein and peptidebiotemplated metal and metal oxide nanoparticles and theirpatterning onto surfacesrdquo Journal of Materials Chemistry vol22 no 25 pp 12423ndash12434 2012

[11] P Mohanpuria N K Rana and S K Yadav ldquoBiosynthesis ofnanoparticles technological concepts and future applicationsrdquoJournal of Nanoparticle Research vol 10 no 3 pp 507ndash517 2008

[12] S K Das and E Marsili ldquoA green chemical approach for thesynthesis of gold nanoparticles characterization andmechanis-tic aspectrdquo Reviews in Environmental Science and Biotechnologyvol 9 no 3 pp 199ndash204 2010

[13] I Sondi and B Salopek-Sondi ldquoSilver nanoparticles as antimi-crobial agent a case study on E coli as a model for Gram-negative bacteriardquo Journal of Colloid and Interface Science vol275 no 1 pp 177ndash182 2004

[14] S-H Kim H-S Lee D-S Ryu S-J Choi and D-S LeeldquoAntibacterial activity of silver-nanoparticles against Staphylo-coccus aureus and Escherichia colirdquo Korean Journal of Microbiol-ogy and Biotechnology vol 39 no 1 pp 77ndash85 2011

BioMed Research International 9

[15] A J Kora and J Arunachalam ldquoAssessment of antibacterialactivity of silver nanoparticles on Pseudomonas aeruginosa andits mechanism of actionrdquo World Journal of Microbiology andBiotechnology vol 27 no 5 pp 1209ndash1216 2011

[16] J S Kim E Kuk K N Yu et al ldquoAntimicrobial effects of silvernanoparticlesrdquo Nanomedicine vol 3 no 1 pp 95ndash101 2007

[17] U K Parashar V Kumar T Bera et al ldquoStudy of mechanismof enhanced antibacterial activity by green synthesis of silvernanoparticlesrdquo Nanotechnology vol 22 no 41 Article ID415104 13 pages 2011

[18] S Bonde ldquoA Biogenic approach for green synthesis of silvernanoparticles using extract of Foeniculum vulgare and itsactivity against S aureus and Ecolirdquo Bioscience vol 3 no 1 pp59ndash63 2011

[19] J Hiremath V Rathod S Ninganagouda D Singh and KPrema ldquoAntibacterial activity of Silver Nanoparticles from Rhi-zopus spp against Gram negative EcoliMDR strainsrdquo Journal ofPure and Applied Microbiology vol 8 no 1 pp 555ndash562 2014

[20] Y Zhou Y Kong S Kundu J D Cirillo and H LiangldquoAntibacterial activities of gold and silver nanoparticles againstEscherichia coli and bacillus Calmette-Guerinrdquo Journal ofNanobiotechnology vol 10 article 19 2012

[21] W K Jung H C Koo K W Kim S Shin S H Kim and YH Park ldquoAntibacterial activity and mechanism of action of thesilver ion in Staphylococcus aureus and Escherichia colirdquoAppliedand Environmental Microbiology vol 74 no 7 pp 2171ndash21782008

[22] M Yamanaka K Hara and J Kudo ldquoBactericidal actions ofa silver ion solution on Escherichia coli studied by energy-filtering transmission electron microscopy and proteomic anal-ysisrdquoApplied and EnvironmentalMicrobiology vol 71 no 11 pp7589ndash7593 2005

[23] Q L Feng J Wu G Q Chen T N Kim and J O Kim ldquoAmechanistic study of the antibacterial effect of silver ions onEscherichia coli and Staphylococcus aureusrdquo Journal of Biomedi-cal Materials Research vol 52 no 4 pp 662ndash668 2000

[24] J R Morones J L Elechiguerra A Camacho et al ldquoThebactericidal effect of silver nanoparticlesrdquo Nanotechnology vol16 no 10 pp 2346ndash2353 2005

[25] Y Matsumura K Yoshikata S-I Kunisaki and T TsuchidoldquoMode of bactericidal action of silver zeolite and its comparisonwith that of silver nitraterdquo Applied and Environmental Microbi-ology vol 69 no 7 pp 4278ndash4281 2003

[26] J A Lemire J JHarrison andR J Turner ldquoAntimicrobial activ-ity of metals mechanisms molecular targets and applicationsrdquoNature Reviews Microbiology vol 11 no 6 pp 371ndash384 2013

[27] AGrigorEva I Saranina N Tikunova et al ldquoFinemechanismsof the interaction of silver nanoparticles with the cells ofSalmonella typhimurium and Staphylococcus aureusrdquo BioMetalsvol 26 no 3 pp 479ndash488 2013

[28] M Chen Z Yang H Wu X Pan X Xie and C WuldquoAntimicrobial activity and the mechanism of silver nanoparti-cle thermosensitive gelrdquo International Journal of Nanomedicinevol 6 pp 2873ndash2877 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 7: Research Article Growth Kinetics and Mechanistic Action of ...downloads.hindawi.com/journals/bmri/2014/753419.pdf · Species Released by Silver Nanoparticles from Aspergillus niger

BioMed Research International 7

2047

minus2048

327640 (mT)

(A) 337475 g = 200025

337640 (mT) 347640 (mT)

(m1) 332715 g = 202887(m2 minus m1) = 87037 (mT)

(m2) 341418 g = 197715

Figure 10 ESR spectrum of AgNPs recorded at room temperaturem1

(332715) and m2

(341418) indicates the control peak of Mnand the peak (mT 337475) indicates the released free radical fromAgNPs Instrument setting JEOL-JES-TE 200 spectrophotometermicropower 4mW MOD 100 khz and the time const 003 sec

20E+06

15E+06

10E+06

50E+05

0

500 600 700 800

Wavelength (nm)

Emission acquistion

Inte

nsity

(cps

)

File 1 = VRSRJ01

Figure 11 Formation of ROS in E coli using fluorescence spec-troscopy

(LPS) molecules and membrane proteins A bacterial mem-brane with this morphology exhibits a significant increase inpermeability leaving the bacterial cells incapable of properlyregulating transport through the plasma membrane andfinally causing cell death It is well known that the outermembrane of E coli cells is predominantly constructed fromtightly packed LPS molecules which provide an effectivepermeability barrier [13] It has been also proposed that thesites of interaction for AgNPs and membrane cells might bedue to sulfur containing proteins in a similar way as silverinteracts with thiol groups of respiratory chain proteins andtransport proteins interfering with their proper function [21ndash25]

Reactive Oxygen Species (ROS) are natural byproducts ofthemetabolismof respiring organisms [16] Induction of ROSsynthesis leads to the formation of highly reactive radicalsthat destroy the cells The bacterial growth inhibition byformation of free radicals from the surface of AgNPs was alsoobserved by employing ESR spectroscopy (Figure 10) Excessgeneration of reactive oxygen species can attack membranelipids and then lead to a breakdown of membrane functionCertain transition metals might disrupt the cellular donorligands that coordinate Fe Mounting evidence suggests that

the primary targets for variousmetals are the solvent exposed[4Fe-4S] clusters of proteins The direct or indirect destruc-tion of [4Fe-4S] clusters by metals could result in the releaseof additional Fenton-active Fe into the cytoplasm resultingin increased ROS formation The ability to induce Fe releasefrom these proteins as well as from other Fe-containingproteins might account for observations that some Fenton-inactive metals (such as Ag Hg and Ga) generate ROS andthat cells require or upregulate ROS-detoxification enzymesto withstand toxic doses of these nanoparticles [26]The ROSproduction from the surface of AgNPs was measured usingDCFH-DAmethod Dichlorofluorescein diacetate (DCFDA)is a popular fluorescence-based probe for reactive oxygenspecies (ROS) detection in vitro After 5 h of incubationROS formed in the sample was detected at 523 nm of emis-sion wavelength using Fluorescence spectroscopy (Figure 11)Intracellular esterases cleave DCFH-DA at the two esterbonds producing a relatively polar and cell membrane-impermeable product H2DCFThis nonfluorescentmoleculeaccumulates intracellularly and subsequent oxidation yieldsthe highly fluorescent product DCF The redox state of thesample can bemonitored by detecting the increase in fluores-cence The result confirms the generation of free radicals andfrom the surface of AgNPs which becomes toxic to bacterialcells leading to death Our result corroborates with Kim etal [14] who reported the oxidative stress can cause damageto bacterial cell membrane protein structure and intracel-luluar system against S aureus and E coli usingDCFDAmethod

To determine the involvement of ROS in the antibacterialactivity of AgNPs we used ascorbic acid as scavenger Thisantioxidant was used to scavenge the ROS produced bythe AgNPs Protective activity of antioxidant against bac-tericidal activity of AgNPs was observed in Figure 12 Incontrol plate the bacterial colonies were clearly seen withoutantioxidants and AgNPs but in the plate supplementedwith AgNPs (10 120583gmL) no bacterial growth was observedrevealing that AgNPs completely inhibited bacterial growthdue to ROS formation Surprisingly the bacterial colonieswere observed in the plate supplemented with both AgNPsand antioxidant which clearly indicates that the ascorbicacid used as an antioxidant serves as a scavenger hinderingthe ROS release from AgNPs It is determined that theantioxidant prevents the formation of a silver oxide layeron the AgNPs surface and consequently formation of theAg+ reservoir Kora and Arunachalam [15] reported the100 P aeruginosa growth survival by using ascorbic acidas scavenger while 73 of bacteria protected from NAC asscavenger

Bacterial cells exposed to AgNPs suffer morphologicalchanges such as cytoplasm shrinkage detachment of cellwall membrane DNA condensation and localization in anelectron-light region in the centre of the cell and cellmembrane degradation allowing the leakage of intracellularcontents [14 27] Physiological changes occur together withthe morphological changes bacterial cells enter an activebut nonculturable state in which physiological levels can bemeasured but cells are not able to grow and replicate [9 28]

8 BioMed Research International

Control SNPs SNPs + ascorbic acid

Figure 12 Antioxidant activities of silver nanoparticles on E coli

4 Conclusion

From our results two aspects can be elucidated one is thefact that AgNPs themselves having antibacterial propertiesact on the pathogenic bacteria by anchoring to the cell surfacetrying to get inside the bacterial cell through ruptured cellmembrane leading to the perforation of cell membrane bycausing leakage of cell components and finally cell deathThesecond aspect is the release of ROS from AgNPs producedby A niger which is clearly evident from the facts thatthe antioxidant ascorbic acid is used as a scavenger ROSproductionwas confirmed by usingDCFH-DAmethodusingfluorescence spectroscopy Antioxidant will inhibit ROS pro-duction thereby luxurious growth of E coli was observedNutrient agar plate inoculated with E coli supplemented withAgNPs without addition of antioxidant showed completeinhibition of E coli suggesting that ROS is released fromAgNPs which completely inhibit E coli Our studies suggestthat 10 120583gmL of AgNPs are sufficient for complete inhibitionof E coli which is a boon to biotechnologists to go deep formechanism studies

Conflict of Interests

The authors report no conflict of interests

Acknowledgments

The Authors would like to thank the Department of Micro-biology Gulbarga University Gulbarga India for provid-ing logistic support and facilities They also thank SAIF-STIC Cochin Ruska labs Hyderabad SAIF-IIT Madras andMalvern Instruments Ltd Bangalore India for characteri-zation studies

References

[1] CMarambio-Jones and EM VHoek ldquoA review of the antibac-terial effects of silver nanomaterials and potential implicationsfor human health and the environmentrdquo Journal of NanoparticleResearch vol 12 no 5 pp 1531ndash1551 2010

[2] D Rejeski ldquoNanotechnology and consumer productsrdquohttpwwwnanotechprojectorgpublicationsarchivenano-technology consumer products

[3] D Singh V Rathod N Shivaraj J Hiremath A K Singhand J Methew ldquoOptimization and characterization of silvernanoparticles by endophytic fungi Penicillium sp isolated from

Curcuma longa (turmeric) and Application studies againstMDR E coli and S aureusrdquo Bioinorganic Chemistry and Appli-cations vol 2014 Article ID 408021 8 pages 2014

[4] A Banu V Rathod and E Ranganath ldquoSilver nanoparticleproduction by Rhizopus stolonifer and its antibacterial activ-ity against extended spectrum 120573-lactamase producing (ESBL)strains of Enterobacteriaceaerdquo Materials Research Bulletin vol46 no 9 pp 1417ndash1423 2011

[5] D Singh V Rathod N Shivaraj J Hiremath and P Kulka-rni ldquoBiosynthesis of silver nanoparticles by endophytic fungiPenicillium sp isolated from Curcuma longa (turmeric) and itsantibacterial activity against pathogenic gram negative bacte-riardquo Journal of Pharmacy Research vol 7 no 5 pp 448ndash4532013

[6] V Rathod A Banu and E Ranganath ldquoBiosynthesis of highlystabilized silver nanoparticles by Rhizopus stolonifer and theirAnti-fungal efficacyrdquo International Journal of Molecular andClinical Microbiology vol 1 no 2 pp 65ndash70 2011

[7] S Galdiero A Falanga M Vitiello M Cantisani V Marra andM Galdiero ldquoSilver nanoparticles as potential antiviral agentsrdquoMolecules vol 16 no 10 pp 8894ndash8918 2011

[8] Y Wang L Cao S Guan et al ldquoSilver mineralization onself-assembled peptide nanofibers for long term antimicrobialeffectrdquo Journal of Materials Chemistry vol 22 no 6 pp 2575ndash2581 2012

[9] W-R Li X-B Xie Q-S Shi H-Y Zeng Y-S Ou-Yang andY-B Chen ldquoAntibacterial activity and mechanism of silvernanoparticles on Escherichia colirdquo Applied Microbiology andBiotechnology vol 85 no 4 pp 1115ndash1122 2010

[10] J M Galloway and S S Staniland ldquoProtein and peptidebiotemplated metal and metal oxide nanoparticles and theirpatterning onto surfacesrdquo Journal of Materials Chemistry vol22 no 25 pp 12423ndash12434 2012

[11] P Mohanpuria N K Rana and S K Yadav ldquoBiosynthesis ofnanoparticles technological concepts and future applicationsrdquoJournal of Nanoparticle Research vol 10 no 3 pp 507ndash517 2008

[12] S K Das and E Marsili ldquoA green chemical approach for thesynthesis of gold nanoparticles characterization andmechanis-tic aspectrdquo Reviews in Environmental Science and Biotechnologyvol 9 no 3 pp 199ndash204 2010

[13] I Sondi and B Salopek-Sondi ldquoSilver nanoparticles as antimi-crobial agent a case study on E coli as a model for Gram-negative bacteriardquo Journal of Colloid and Interface Science vol275 no 1 pp 177ndash182 2004

[14] S-H Kim H-S Lee D-S Ryu S-J Choi and D-S LeeldquoAntibacterial activity of silver-nanoparticles against Staphylo-coccus aureus and Escherichia colirdquo Korean Journal of Microbiol-ogy and Biotechnology vol 39 no 1 pp 77ndash85 2011

BioMed Research International 9

[15] A J Kora and J Arunachalam ldquoAssessment of antibacterialactivity of silver nanoparticles on Pseudomonas aeruginosa andits mechanism of actionrdquo World Journal of Microbiology andBiotechnology vol 27 no 5 pp 1209ndash1216 2011

[16] J S Kim E Kuk K N Yu et al ldquoAntimicrobial effects of silvernanoparticlesrdquo Nanomedicine vol 3 no 1 pp 95ndash101 2007

[17] U K Parashar V Kumar T Bera et al ldquoStudy of mechanismof enhanced antibacterial activity by green synthesis of silvernanoparticlesrdquo Nanotechnology vol 22 no 41 Article ID415104 13 pages 2011

[18] S Bonde ldquoA Biogenic approach for green synthesis of silvernanoparticles using extract of Foeniculum vulgare and itsactivity against S aureus and Ecolirdquo Bioscience vol 3 no 1 pp59ndash63 2011

[19] J Hiremath V Rathod S Ninganagouda D Singh and KPrema ldquoAntibacterial activity of Silver Nanoparticles from Rhi-zopus spp against Gram negative EcoliMDR strainsrdquo Journal ofPure and Applied Microbiology vol 8 no 1 pp 555ndash562 2014

[20] Y Zhou Y Kong S Kundu J D Cirillo and H LiangldquoAntibacterial activities of gold and silver nanoparticles againstEscherichia coli and bacillus Calmette-Guerinrdquo Journal ofNanobiotechnology vol 10 article 19 2012

[21] W K Jung H C Koo K W Kim S Shin S H Kim and YH Park ldquoAntibacterial activity and mechanism of action of thesilver ion in Staphylococcus aureus and Escherichia colirdquoAppliedand Environmental Microbiology vol 74 no 7 pp 2171ndash21782008

[22] M Yamanaka K Hara and J Kudo ldquoBactericidal actions ofa silver ion solution on Escherichia coli studied by energy-filtering transmission electron microscopy and proteomic anal-ysisrdquoApplied and EnvironmentalMicrobiology vol 71 no 11 pp7589ndash7593 2005

[23] Q L Feng J Wu G Q Chen T N Kim and J O Kim ldquoAmechanistic study of the antibacterial effect of silver ions onEscherichia coli and Staphylococcus aureusrdquo Journal of Biomedi-cal Materials Research vol 52 no 4 pp 662ndash668 2000

[24] J R Morones J L Elechiguerra A Camacho et al ldquoThebactericidal effect of silver nanoparticlesrdquo Nanotechnology vol16 no 10 pp 2346ndash2353 2005

[25] Y Matsumura K Yoshikata S-I Kunisaki and T TsuchidoldquoMode of bactericidal action of silver zeolite and its comparisonwith that of silver nitraterdquo Applied and Environmental Microbi-ology vol 69 no 7 pp 4278ndash4281 2003

[26] J A Lemire J JHarrison andR J Turner ldquoAntimicrobial activ-ity of metals mechanisms molecular targets and applicationsrdquoNature Reviews Microbiology vol 11 no 6 pp 371ndash384 2013

[27] AGrigorEva I Saranina N Tikunova et al ldquoFinemechanismsof the interaction of silver nanoparticles with the cells ofSalmonella typhimurium and Staphylococcus aureusrdquo BioMetalsvol 26 no 3 pp 479ndash488 2013

[28] M Chen Z Yang H Wu X Pan X Xie and C WuldquoAntimicrobial activity and the mechanism of silver nanoparti-cle thermosensitive gelrdquo International Journal of Nanomedicinevol 6 pp 2873ndash2877 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 8: Research Article Growth Kinetics and Mechanistic Action of ...downloads.hindawi.com/journals/bmri/2014/753419.pdf · Species Released by Silver Nanoparticles from Aspergillus niger

8 BioMed Research International

Control SNPs SNPs + ascorbic acid

Figure 12 Antioxidant activities of silver nanoparticles on E coli

4 Conclusion

From our results two aspects can be elucidated one is thefact that AgNPs themselves having antibacterial propertiesact on the pathogenic bacteria by anchoring to the cell surfacetrying to get inside the bacterial cell through ruptured cellmembrane leading to the perforation of cell membrane bycausing leakage of cell components and finally cell deathThesecond aspect is the release of ROS from AgNPs producedby A niger which is clearly evident from the facts thatthe antioxidant ascorbic acid is used as a scavenger ROSproductionwas confirmed by usingDCFH-DAmethodusingfluorescence spectroscopy Antioxidant will inhibit ROS pro-duction thereby luxurious growth of E coli was observedNutrient agar plate inoculated with E coli supplemented withAgNPs without addition of antioxidant showed completeinhibition of E coli suggesting that ROS is released fromAgNPs which completely inhibit E coli Our studies suggestthat 10 120583gmL of AgNPs are sufficient for complete inhibitionof E coli which is a boon to biotechnologists to go deep formechanism studies

Conflict of Interests

The authors report no conflict of interests

Acknowledgments

The Authors would like to thank the Department of Micro-biology Gulbarga University Gulbarga India for provid-ing logistic support and facilities They also thank SAIF-STIC Cochin Ruska labs Hyderabad SAIF-IIT Madras andMalvern Instruments Ltd Bangalore India for characteri-zation studies

References

[1] CMarambio-Jones and EM VHoek ldquoA review of the antibac-terial effects of silver nanomaterials and potential implicationsfor human health and the environmentrdquo Journal of NanoparticleResearch vol 12 no 5 pp 1531ndash1551 2010

[2] D Rejeski ldquoNanotechnology and consumer productsrdquohttpwwwnanotechprojectorgpublicationsarchivenano-technology consumer products

[3] D Singh V Rathod N Shivaraj J Hiremath A K Singhand J Methew ldquoOptimization and characterization of silvernanoparticles by endophytic fungi Penicillium sp isolated from

Curcuma longa (turmeric) and Application studies againstMDR E coli and S aureusrdquo Bioinorganic Chemistry and Appli-cations vol 2014 Article ID 408021 8 pages 2014

[4] A Banu V Rathod and E Ranganath ldquoSilver nanoparticleproduction by Rhizopus stolonifer and its antibacterial activ-ity against extended spectrum 120573-lactamase producing (ESBL)strains of Enterobacteriaceaerdquo Materials Research Bulletin vol46 no 9 pp 1417ndash1423 2011

[5] D Singh V Rathod N Shivaraj J Hiremath and P Kulka-rni ldquoBiosynthesis of silver nanoparticles by endophytic fungiPenicillium sp isolated from Curcuma longa (turmeric) and itsantibacterial activity against pathogenic gram negative bacte-riardquo Journal of Pharmacy Research vol 7 no 5 pp 448ndash4532013

[6] V Rathod A Banu and E Ranganath ldquoBiosynthesis of highlystabilized silver nanoparticles by Rhizopus stolonifer and theirAnti-fungal efficacyrdquo International Journal of Molecular andClinical Microbiology vol 1 no 2 pp 65ndash70 2011

[7] S Galdiero A Falanga M Vitiello M Cantisani V Marra andM Galdiero ldquoSilver nanoparticles as potential antiviral agentsrdquoMolecules vol 16 no 10 pp 8894ndash8918 2011

[8] Y Wang L Cao S Guan et al ldquoSilver mineralization onself-assembled peptide nanofibers for long term antimicrobialeffectrdquo Journal of Materials Chemistry vol 22 no 6 pp 2575ndash2581 2012

[9] W-R Li X-B Xie Q-S Shi H-Y Zeng Y-S Ou-Yang andY-B Chen ldquoAntibacterial activity and mechanism of silvernanoparticles on Escherichia colirdquo Applied Microbiology andBiotechnology vol 85 no 4 pp 1115ndash1122 2010

[10] J M Galloway and S S Staniland ldquoProtein and peptidebiotemplated metal and metal oxide nanoparticles and theirpatterning onto surfacesrdquo Journal of Materials Chemistry vol22 no 25 pp 12423ndash12434 2012

[11] P Mohanpuria N K Rana and S K Yadav ldquoBiosynthesis ofnanoparticles technological concepts and future applicationsrdquoJournal of Nanoparticle Research vol 10 no 3 pp 507ndash517 2008

[12] S K Das and E Marsili ldquoA green chemical approach for thesynthesis of gold nanoparticles characterization andmechanis-tic aspectrdquo Reviews in Environmental Science and Biotechnologyvol 9 no 3 pp 199ndash204 2010

[13] I Sondi and B Salopek-Sondi ldquoSilver nanoparticles as antimi-crobial agent a case study on E coli as a model for Gram-negative bacteriardquo Journal of Colloid and Interface Science vol275 no 1 pp 177ndash182 2004

[14] S-H Kim H-S Lee D-S Ryu S-J Choi and D-S LeeldquoAntibacterial activity of silver-nanoparticles against Staphylo-coccus aureus and Escherichia colirdquo Korean Journal of Microbiol-ogy and Biotechnology vol 39 no 1 pp 77ndash85 2011

BioMed Research International 9

[15] A J Kora and J Arunachalam ldquoAssessment of antibacterialactivity of silver nanoparticles on Pseudomonas aeruginosa andits mechanism of actionrdquo World Journal of Microbiology andBiotechnology vol 27 no 5 pp 1209ndash1216 2011

[16] J S Kim E Kuk K N Yu et al ldquoAntimicrobial effects of silvernanoparticlesrdquo Nanomedicine vol 3 no 1 pp 95ndash101 2007

[17] U K Parashar V Kumar T Bera et al ldquoStudy of mechanismof enhanced antibacterial activity by green synthesis of silvernanoparticlesrdquo Nanotechnology vol 22 no 41 Article ID415104 13 pages 2011

[18] S Bonde ldquoA Biogenic approach for green synthesis of silvernanoparticles using extract of Foeniculum vulgare and itsactivity against S aureus and Ecolirdquo Bioscience vol 3 no 1 pp59ndash63 2011

[19] J Hiremath V Rathod S Ninganagouda D Singh and KPrema ldquoAntibacterial activity of Silver Nanoparticles from Rhi-zopus spp against Gram negative EcoliMDR strainsrdquo Journal ofPure and Applied Microbiology vol 8 no 1 pp 555ndash562 2014

[20] Y Zhou Y Kong S Kundu J D Cirillo and H LiangldquoAntibacterial activities of gold and silver nanoparticles againstEscherichia coli and bacillus Calmette-Guerinrdquo Journal ofNanobiotechnology vol 10 article 19 2012

[21] W K Jung H C Koo K W Kim S Shin S H Kim and YH Park ldquoAntibacterial activity and mechanism of action of thesilver ion in Staphylococcus aureus and Escherichia colirdquoAppliedand Environmental Microbiology vol 74 no 7 pp 2171ndash21782008

[22] M Yamanaka K Hara and J Kudo ldquoBactericidal actions ofa silver ion solution on Escherichia coli studied by energy-filtering transmission electron microscopy and proteomic anal-ysisrdquoApplied and EnvironmentalMicrobiology vol 71 no 11 pp7589ndash7593 2005

[23] Q L Feng J Wu G Q Chen T N Kim and J O Kim ldquoAmechanistic study of the antibacterial effect of silver ions onEscherichia coli and Staphylococcus aureusrdquo Journal of Biomedi-cal Materials Research vol 52 no 4 pp 662ndash668 2000

[24] J R Morones J L Elechiguerra A Camacho et al ldquoThebactericidal effect of silver nanoparticlesrdquo Nanotechnology vol16 no 10 pp 2346ndash2353 2005

[25] Y Matsumura K Yoshikata S-I Kunisaki and T TsuchidoldquoMode of bactericidal action of silver zeolite and its comparisonwith that of silver nitraterdquo Applied and Environmental Microbi-ology vol 69 no 7 pp 4278ndash4281 2003

[26] J A Lemire J JHarrison andR J Turner ldquoAntimicrobial activ-ity of metals mechanisms molecular targets and applicationsrdquoNature Reviews Microbiology vol 11 no 6 pp 371ndash384 2013

[27] AGrigorEva I Saranina N Tikunova et al ldquoFinemechanismsof the interaction of silver nanoparticles with the cells ofSalmonella typhimurium and Staphylococcus aureusrdquo BioMetalsvol 26 no 3 pp 479ndash488 2013

[28] M Chen Z Yang H Wu X Pan X Xie and C WuldquoAntimicrobial activity and the mechanism of silver nanoparti-cle thermosensitive gelrdquo International Journal of Nanomedicinevol 6 pp 2873ndash2877 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 9: Research Article Growth Kinetics and Mechanistic Action of ...downloads.hindawi.com/journals/bmri/2014/753419.pdf · Species Released by Silver Nanoparticles from Aspergillus niger

BioMed Research International 9

[15] A J Kora and J Arunachalam ldquoAssessment of antibacterialactivity of silver nanoparticles on Pseudomonas aeruginosa andits mechanism of actionrdquo World Journal of Microbiology andBiotechnology vol 27 no 5 pp 1209ndash1216 2011

[16] J S Kim E Kuk K N Yu et al ldquoAntimicrobial effects of silvernanoparticlesrdquo Nanomedicine vol 3 no 1 pp 95ndash101 2007

[17] U K Parashar V Kumar T Bera et al ldquoStudy of mechanismof enhanced antibacterial activity by green synthesis of silvernanoparticlesrdquo Nanotechnology vol 22 no 41 Article ID415104 13 pages 2011

[18] S Bonde ldquoA Biogenic approach for green synthesis of silvernanoparticles using extract of Foeniculum vulgare and itsactivity against S aureus and Ecolirdquo Bioscience vol 3 no 1 pp59ndash63 2011

[19] J Hiremath V Rathod S Ninganagouda D Singh and KPrema ldquoAntibacterial activity of Silver Nanoparticles from Rhi-zopus spp against Gram negative EcoliMDR strainsrdquo Journal ofPure and Applied Microbiology vol 8 no 1 pp 555ndash562 2014

[20] Y Zhou Y Kong S Kundu J D Cirillo and H LiangldquoAntibacterial activities of gold and silver nanoparticles againstEscherichia coli and bacillus Calmette-Guerinrdquo Journal ofNanobiotechnology vol 10 article 19 2012

[21] W K Jung H C Koo K W Kim S Shin S H Kim and YH Park ldquoAntibacterial activity and mechanism of action of thesilver ion in Staphylococcus aureus and Escherichia colirdquoAppliedand Environmental Microbiology vol 74 no 7 pp 2171ndash21782008

[22] M Yamanaka K Hara and J Kudo ldquoBactericidal actions ofa silver ion solution on Escherichia coli studied by energy-filtering transmission electron microscopy and proteomic anal-ysisrdquoApplied and EnvironmentalMicrobiology vol 71 no 11 pp7589ndash7593 2005

[23] Q L Feng J Wu G Q Chen T N Kim and J O Kim ldquoAmechanistic study of the antibacterial effect of silver ions onEscherichia coli and Staphylococcus aureusrdquo Journal of Biomedi-cal Materials Research vol 52 no 4 pp 662ndash668 2000

[24] J R Morones J L Elechiguerra A Camacho et al ldquoThebactericidal effect of silver nanoparticlesrdquo Nanotechnology vol16 no 10 pp 2346ndash2353 2005

[25] Y Matsumura K Yoshikata S-I Kunisaki and T TsuchidoldquoMode of bactericidal action of silver zeolite and its comparisonwith that of silver nitraterdquo Applied and Environmental Microbi-ology vol 69 no 7 pp 4278ndash4281 2003

[26] J A Lemire J JHarrison andR J Turner ldquoAntimicrobial activ-ity of metals mechanisms molecular targets and applicationsrdquoNature Reviews Microbiology vol 11 no 6 pp 371ndash384 2013

[27] AGrigorEva I Saranina N Tikunova et al ldquoFinemechanismsof the interaction of silver nanoparticles with the cells ofSalmonella typhimurium and Staphylococcus aureusrdquo BioMetalsvol 26 no 3 pp 479ndash488 2013

[28] M Chen Z Yang H Wu X Pan X Xie and C WuldquoAntimicrobial activity and the mechanism of silver nanoparti-cle thermosensitive gelrdquo International Journal of Nanomedicinevol 6 pp 2873ndash2877 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Page 10: Research Article Growth Kinetics and Mechanistic Action of ...downloads.hindawi.com/journals/bmri/2014/753419.pdf · Species Released by Silver Nanoparticles from Aspergillus niger

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of