Research Article Screening Antimicrobial Activity of …downloads.hindawi.com/journals/jnp/2016/4694367.pdfResearch Article Screening Antimicrobial Activity of Nickel Nanoparticles

Research ArticleScreening Antimicrobial Activity of Nickel NanoparticlesSynthesized Using Ocimum sanctum Leaf Extract

Chitra Jeyaraj Pandian1 Rameshthangam Palanivel2 and Solairaj Dhanasekaran2

1Department of Biochemistry Manonmaniam Sundaranar University Tirunelveli Tamil Nadu 627012 India2Department of Biotechnology Science Campus Alagappa University Karaikudi Tamil Nadu 630 004 India

Correspondence should be addressed to Rameshthangam Palanivel rameshthangamalagappauniversityacin

Received 19 October 2015 Accepted 15 February 2016

Academic Editor Tapas Sen

Copyright copy 2016 Chitra Jeyaraj Pandian 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

Antimicrobial efficacy of nickel nanoparticles synthesized using leaf extract of Ocimum sanctum (NiGs) was investigated againstpathogenic Gram-negative (E coli K pneumoniae and S typhi) Gram-positive (B subtilis S epidermidis) bacteria and fungi (Calbicans C tropicalis A fumigatus A clavatus and A niger) 100120583gmL NiGs showed maximum antimicrobial activity againsttested pathogens compared to leaf extract and antibiotics E coli (25mm) and C albicans (23mm) exhibited higher zone ofinhibition at 100 120583gmL NiGs MIC MBC and MFC values of NiGs against all tested pathogens ranged between 25 and 50 120583gmLGrowth of bacterial and fungal cells (105 cfumL) was completely inhibited at 50 120583gmL NiGs E coli and C albicans have showedstrong antimicrobial activity with 81 and 50 reactive oxygen species (ROS) production 30 and 16120583gmL protein leakageand 95 and 82UL LDH leakages respectively Gram-negative bacteria and Candida species showed more sensitivity to NiGs atall concentrations tested (25ndash100 120583gmL) than Gram-positive bacteria and Aspergillus species respectively Microbial growth inthe presence of NiGs and ascorbic acid confirmed the involvement of ROS in antimicrobial activity Hence NiGs induced ROSgeneration was attributed to the protein and LDH leakage from microbial membranes

1 Introduction

Recent increase in microbial resistance to diverse antibioticsand uncertainties in health care cost lead to the emergenceof more economical new methods to produce nanoparti-cles with specific physical chemical properties and limitedresistance [1] The antimicrobial activities of nanoparticleshave been attributed to their relatively smaller sizes andhigh amount of surface-area-to-volume ratio that facilitateinteracting closely with membranes of viruses fungi andbacteria [2] Antimicrobial activities of metal nanoparticleslike Ag Cu Ni and Co (and their oxides) have beenpreviously reported [3] Stabilizing and protective agentsin chemical synthesis of nanoparticles interact chemicallywith the surface of nickel nanoparticles and modify theirmorphology electronic and magnetic properties [4] Inrecent years there is an increasing emphasis on greensynthesis of metal nanoparticles because of their applica-tion in utilization of nontoxic renewable chemicals and

elimination of generated waste [5] Polymeric nanoparticleslike chitin nanoparticles were also found to be low costbiodegradable material for environmental protection [6] Butthe plant-mediated nanoparticles synthesis is rapid cost-effective ecofriendly and safe single step method for humantherapeutic use [7] Medicinal plant Ocimum sanctum hasbeen found to be highly effective in different types of animalmodels for antimicrobial immunomodulatory antistressanti-inflammatory antipyretic antiasthmatic hypoglycemichypotensive and analgesic activities [8] O sanctum leaveshave been reported to show strong antifungal activities andantibacterial activity [9] Earlier studies provide substantialevidence that nanoparticles produce reactive oxygen species(ROS) in bacterial cells and ROS accumulation intracellularlyregulates apoptosis [10] Oxidative stress-induced respiratorycells damage can be determined by measuring respiratorychain lactate dehydrogenase activity in microbial cells andnanoparticles enhance protein leakage by increasing mem-brane permeability [11] In our previous work [12] we have

Hindawi Publishing CorporationJournal of NanoparticlesVolume 2016 Article ID 4694367 13 pageshttpdxdoiorg10115520164694367

2 Journal of Nanoparticles

synthesized and characterized nickel nanoparticles usingleaf extracts of Ocimum sanctum with 1mM of aqueousnickel nitrate Antimicrobial activity of synthesized nickelnanoparticles was evaluated by various susceptibility assayson bacterial and fungal pathogens The toxicity of NiGs wasstudied against Gram-negative (Escherichia coli Klebsiellapneumoniaeand Salmonella typhi) and Gram-positive (Bacil-lus subtilis Staphylococcus epidermidis) bacterial and fungalpathogens (Candida albicans Candida tropicalis Aspergillusfumigatus Aspergillus clavatusand Aspergillus niger) Wealso substantiate that NiGs induced reactive oxygen speciesformation destroys microbial cell membrane and its perme-ability leading to growth suppression and cell death

2 Materials and Methods

21 Materials and Strains O sanctum leaves were collectedfrom Karaikudi town Tamil Nadu India Taxonomic iden-tification was done by Department of Botany AlagappaUniversity Karaikudi Tamil Nadu India

Lyophilized cultures of Escherichia coli (MTCC 1682)Klebsiella pneumoniae (MTCC 8911) Salmonella typhi(MTCC 3224) Bacillus subtilis (MTCC 6133) Staphylococcusepidermidis (MTCC 7919) Candida albicans (MTCC 3018)Candida tropicalis (MTCC 6222) Aspergillus fumigatus(MTCC 2508) Aspergillus clavatus (MTCC 1323) and Asper-gillus niger (MTCC 281) were procured from Microbial TypeCulture Collection (MTCC) located in Indian Institute ofMicrobial Technology Chandigarh India Mueller-Hintonagar media for bacteria and potato dextrose agar media forfungi were purchased fromHi-Media Laboratories MumbaiIndia Nickel nitrate was purchased from SD Fine ChemicalsLtd Mumbai India All other chemicals were purchasedfrom Sigma-Aldrich Mumbai India

22 Synthesis and Characterization of Nanoparticles Nickelnanoparticles were synthesized using the leaf extract of Osanctum and characterized according to our previous study[12] Chemical interaction of compounds present inOcimumsanctum leaf extract with nanoparticles and morphologyof NiGs were characterized by Fourier transform infrared(FTIR) spectroscopy and transmission electron microscope(TEM) respectively

23 Dose-Dependent Antimicrobial Assay Antibacterial andantifungal assayswere performedwithMueller-Hinton (MH)agar and Sabouraud dextrose (SD) agarmedium respectivelyBacterial and fungal cultures were prepared to 05McFarlandstandards prior to the assay Antimicrobial activity of NiGswas evaluated by disc diffusion assay against the Gram-negative bacteria (E coli K pneumoniae and S typhi)Gram-positive bacteria (B subtilis S epidermidis) and fungi(C albicans C tropicalis A clavatus A fumigatus and Aniger) Pure microbial cultures were subcultured on nutrientagar and uniformly swabbed on individual plates 20120583L ofNiGs at different concentrations (25 50 and 100 120583gmL) wasimpregnated to 6mm filter paper discs dried and placed onthe culture plate Bacterial and fungal cultureswere incubated

at 37∘C for 24 h and 48 h respectively [13] Antimicrobialactivities were studied by the diameter of zone of inhibitionDeionized water (as control) and leaf extract were used tocompare the antimicrobial activity of NiGs

24 Minimum Inhibitory Concentration (MIC) MinimumBactericidal Concentration (MBC) and Minimum Fungici-dal Concentration (MFC) Assay 100 120583L of NiGs in seriallydescending concentrations 200 to 1120583gmL was added tomicrotitre plates with 100120583LMHbroth for bacterial or 100120583LSD broth for fungal assays Dilutions were done by twofoldserial dilution and 100120583L of bacterial and fungal sampleswas inoculated to respective wells Bacterial and fungal plateswere incubated for 24 h and 48 h respectively at 37∘C [13] andoptical densities were determined at 600 nmusingmicroplatereader Antibiotics (amoxicillin (Amx) and nystatin (Nys))were used to compare the bactericidal and fungicidal activityof NiGs

MBC and MFC were determined by subculturing 2 120583Lof above MIC serial dilution after 24 h of incubation inrespective wells containing 100120583L of broth per well Bacterialand fungal colonies were quantified by further incubationfor 24 h and 48 h respectively at 37∘C MBC and MFC werethe lowest concentration of nanoparticles or antibiotics thatprevented the growth of bacterial and fungal colonies on solidmedia respectively [14]

25 Determination of Microbial Growth Kinetics in thePresence of NiGs Microbial growth rate was observed byinoculating the microtitre plates with MH and SD brothcontaining 105 colony forming units (cfu) per mL of bacterialor fungal pathogens respectively and loaded with varyingconcentrations of nanoparticles (0 25 50 and 100 120583gmL)The plates were incubated at 37∘C and shaken at 180 rpmAfter inoculation the optical density (OD) at 600 nm [15]was serially monitored at every 3 h interval till 24 h andevery 6 h interval till 48 h for bacterial and fungal pathogensrespectively

26 Detection of Reactive Oxygen Species (ROS) Reactiveoxygen species produced in microbial cells were determinedby 2101584071015840 dichlorofluorescein diacetate (DCFDA) Intracellularesterase cleaves fluorescence based probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) to polar impermeable non-fluorescent molecule that accumulates intracellularly and itssubsequent oxidation yields highly fluorescent product 2101584071015840dichlorofluorescein (DCF) which was monitored by increasein fluorescence 105 cfumL bacterial and fungal pathogenswere treated with 0 25 50 and 100 120583gmL NiGs and incu-bated at 37∘C for 6 h and 24 h respectively After incubationthe cultures were centrifuged at 5000 rpm for 10min at 4∘CThe supernatant was treated with 100 120583M DCFDA for 1 hand the ROS formed (fluorescence intensity of DCF) wasdetermined at 488 nm excitation wavelength and 535 nmemission wavelength using fluorescence spectrophotometer[11]

Journal of Nanoparticles 3

27 Assaying the Effect of NiGs on Protein Leakage Bradfordmethod was performed to analyze the protein leakage inthe microbial cells The bacterial and fungal pathogens(105 cfumL) were treated with 0 25 50 and 100 120583gmL NiGsfor 6 h and 24 h respectively After incubation the contentswere centrifuged at 5000 rpm for 5min and supernatant wascollected 200 120583L of supernatant from each sample wasmixedwith 800 120583L of Bradford reagent and incubated for 10min indark at 37∘C The optical density was determined at 595 nmwith bovine serum albumin as standard [16]

28 Assaying the Effect of NiGs on Lactate Dehydrogenase(LDH) Activity The cytoplasmic enzyme LDH release andcell membrane instability were studied as per the procedurereported byArokiyaraj et al (2014) [17] 100 120583L of supernatantfrom each microbial culture treated with different concen-trations of NiGs (0 25 50 and 100 120583gmL) was added tothe reaction mixture containing 05mL of 100mM pyruvate5mg NADH in 20mL of 500mM potassium phosphatebuffer and pH 75 at 30∘C Absorbance (119860) was recordedfor 05 to 5min and relative change in the absorbanceper minute (Δ119860min) was calculated at 340 nm using UV-visible spectrophotometer LDH activity was expressed ininternational unit (UL) which is the amount of enzyme thatreduces 1 120583M of NAD per min at specific temperature

UL = Δ119860min times TV times 1000119889

times 120576 times SV (1)

where TV is the total reaction volume 1000 is the conversionof UmL into UL 119889 is the light path in cm 120576 is theabsorptivity of NADH in mM and SV is the sample volumein mL

29 Determining the Effect of Antioxidant on MicrobicidalActivity of NiGs The involvement of free radicals formationby NiGs was confirmed using antioxidant ascorbic acid thatacts as scavenger of free radicals [18] MH and SD broth with105 cfumL bacterial and fungal samples were supplementedwith 100 120583gmL NiGs and 10mM ascorbic acid and wereshaken at 180 rpm at 37∘CThe growth rate at OD 600 nmwasdetermined at regular intervals till 24 h and 72 h of incubationfor bacterial and fungal pathogens respectively

210 Statistical Analysis All the experiments were performedin triplicate and the data were expressed as the mean plusmn stan-dard deviation (SD) Error bars represent standard deviationsof duplicate experiments

3 Results and Discussion

31 Synthesis and Characterization of NiGs NiGs were syn-thesized usingO sanctum leaf extract and the physiochemicalproperties were characterized (FTIR and TEM) and reportedin our previous work [12]

The FTIR spectrum of NiGs in our previous study [12]evidenced the presence of functional groups such as OndashHstretching carboxylic acid CndashN or CndashC triple bond NndashHbendprimary amines CndashC stretch aliphatic amines andCndashN

Table 1 Zone of inhibition (mean plusmn SD) of NiGs against bacterialand fungal pathogens

Zone of inhibition (mm)

Bacteria Leaf extract NiGs (120583gmL)25 50 100

E coli 90 plusmn 02 153 plusmn 03 192 plusmn 05 251 plusmn 01K pneumoniae 78 plusmn 09 95 plusmn 05 181 plusmn 07 233 plusmn 07S typhi 58 plusmn 03 80 plusmn 14 183 plusmn 03 202 plusmn 03B subtilis 52 plusmn 05 71 plusmn 08 165 plusmn 02 195 plusmn 05S epidermidis 39 plusmn 06 52 plusmn 03 160 plusmn 08 186 plusmn 09

Fungi Leaf extract NiGs (120583gmL)25 50 100

C albicans 78 plusmn 01 145 plusmn 04 182 plusmn 07 231 plusmn 04C tropicalis 52 plusmn 06 124 plusmn 08 144 plusmn 02 216 plusmn 09A clavatus 45 plusmn 03 104 plusmn 01 143 plusmn 01 187 plusmn 05A fumigatus 37 plusmn 04 85 plusmn 03 107 plusmn 09 155 plusmn 06A niger 25 plusmn 01 62 plusmn 05 75 plusmn 05 124 plusmn 01

rock in alkanes indicating the interaction of metabolites andproteins in the O sanctum leaf extract with the NiGs Theseinteracting biological molecules could have been involved inthe formation and stabilization of NiGs in aqueous mediumThis is in concurrence with the findings by Mallikarjunaet al [19] who have reported that the amino acid residuesor proteins in O sanctum leaf extract can strongly bind tosilver nanoparticles preventing its agglomeration and hencestabilizing the nanoparticles

TEM micrograph of the NiGs in our previous study [12]showed that the shape of the NiGs was almost spherical andthe distribution of the particles was narrow The diameterof the sample ranged between 12 and 36 nm and shows acertain extent of particles agglomeration [12] Our study isin good agreement with previous study by Govindasamy etal [20] and Wang et al [21] who have reported that thevaried shape (irregular polygonal cylindrical and spherical)of nickel nanoparticles with particle agglomeration is dueto magnetic interaction and polymer adherence between theparticles respectively

32 Antibacterial and Antifungal Activity of NiGs In discdiffusion assay the suppression of bacterial growth wasdetermined in Petri plates loaded with 25ndash100 120583gmL NiGsafter 24 h at 37∘C (Figure 1(a) and Table 1) Zone of inhibitionwas not observed in control plates loaded with deionizedwater while leaf extract has showed higher growth inhibitionDiameter of inhibition zone ranged from 153 to 251mm 95to 233mm 8 to 202mm 71 to 195mm and 52 to 186mm inE coli K pneumoniae S typhi B subtilis and S epidermidisrespectively with increase in NiGs concentration from 25 to100 120583gmL It is evident fromFigure 1(a) that size of inhibitionzone increases linearly with increase in NiGs concentration(25ndash100 120583gmL)

The presence of inhibition zone in Figure 1(b) confirmedthe antifungal activity of NiGs The size of inhibition zonediffered with the type of fungal pathogen and concentra-tion of NiGs Maximum antifungal activity was observed


(1) Control(2) Leaf extract

EC E coliKP K pneumoniae ST S typhi BS B subtilis SE S epidermidis

12

4

3

5

SEBS

3

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14

5

3KP

4

2

1

5

EC

2

5

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45

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31

4

ST

(5) 20120583gmL NiGs(4) 10120583gmL NiGs(3) 5120583gmL NiGs

(a)


CA C albicansCT C tropicalisAF A fumigates AC A clavatus AN A niger

12

4

3

5

CA

1

5

2

4

3

CT3

5

4

21

AF

1 23

4

5

ANAC

3

2

1

5

4


(b)

Figure 1 Zone of inhibition of (a) bacterial and (b) fungal pathogens treated with NiGs


Table 2 MIC MBC and MFC (mean plusmn SD) of NiGs against bacterial and fungal pathogens

Bacteria Leaf extract (120583gmL) Amoxicillin (120583gmL) NiGs (120583gmL)MIC MBC MIC MBC MIC MBC

E coli 50 plusmn 03 50 plusmn 01 25 plusmn 03 50 plusmn 13 25 plusmn 03 25 plusmn 24K pneumoniae 50 plusmn 01 50 plusmn 00 50 plusmn 20 50 plusmn 01 25 plusmn 28 25 plusmn 07S typhi 50 plusmn 10 100 plusmn 08 50 plusmn 04 100 plusmn 00 25 plusmn 02 25 plusmn 16B subtilis 100 plusmn 05 100 plusmn 13 50 plusmn 17 100 plusmn 02 50 plusmn 02 50 plusmn 02S epidermidis 100 plusmn 02 100 plusmn 06 50 plusmn 02 100 plusmn 01 50 plusmn 05 50 plusmn 01

Fungi Leaf extract (120583gmL) Nystatin (120583gmL) NiGs (120583gmL)MIC MFC MIC MFC MIC MFC

C albicans 50 plusmn 02 50 plusmn 04 50 plusmn 01 50 plusmn 21 25 plusmn 14 50 plusmn 00C tropicalis 50 plusmn 01 100 plusmn 01 50 plusmn 07 100 plusmn 08 25 plusmn 07 50 plusmn 01A clavatus 100 plusmn 25 100 plusmn 09 50 plusmn 00 100 plusmn 12 50 plusmn 25 50 plusmn 05A fumigatus 100 plusmn 00 100 plusmn 13 100 plusmn 03 100 plusmn 00 50 plusmn 10 50 plusmn 12A niger 100 plusmn 09 100 plusmn 02 100 plusmn 10 100 plusmn 03 50 plusmn 08 50 plusmn 06

at 100 120583gmL NiGs for C albicans (231mm) C tropicalis(216mm) A clavatus (187mm) A fumigatus (155mm)and A niger (124mm) Zone of inhibition was not observedin plates loaded with deionized water for tested fungalpathogens Leaf extract showed minimal antifungal activitywith inhibition zone diameter of 78 52 45 37 and 25mmfor C albicans C tropicalis A clavatus A fumigatus andA niger respectively Maximum antimicrobial activity wasobserved at 100 120583gmL NiGs for all tested pathogens at lowerconcentrations (25 50120583gmL) Therefore it is clear from thedata that the antibacterial and antifungal activities are dose-dependent

Disc diffusion study of NiGs confirmed that bacterialand fungal growth inhibition was dose-dependent withmaximum activity at 100 120583gmL and it could be due to therelease of nickel ions from NiGs that increased membranepermeability and ROS generation leading to cell deathThis observation is in good agreement with the earlierantibacterial reports for zinc oxide nanoparticles at 2 to12mM concentration [22] and for silver nanoparticles atconcentration 10 to 150 120583M [14 23]

33 Minimum Inhibitory Concentration (MIC) and MinimumMicrobicidal Concentration (MBCMFC) MIC of NiGs wasstudied to determine the lowest concentration that couldcompletely inhibit visible growth of bacterial and fungalpathogens Antimicrobial activity of NiGs in terms of MICMBC and MFC is shown in Table 2 Both inhibitory andbactericidal concentrations of leaf extract were found tobe 100 120583gmL for Gram-positive bacteria (B subtilis and Sepidermidis) and Aspergillus species (A clavatus A fumiga-tus and A niger) while 50 120583gmL was observed for Gram-negative bacteria (E coli K pneumonia and S typhi) andCandida species (C albicans and C tropicalis)

25 120583gmLNiGs were the minimum inhibitory concentra-tion against all bacterial pathogens 25 120583gmL NiGs showed

bactericidal activity against E coliK pneumonia and S typhiwhile 50120583gmL NiGs exhibited bactericidal activity againstB subtilis and S epidermidis This data is comparable withthe antibiotic amoxicillin which showed inhibitory activity at50 120583gmL for all bacterial pathogens and bactericidal activityof 100120583gmL for S typhi B subtilis and S epidermidis25 120583gmL amoxicillin has showed bactericidal activity againstE coli K pneumoniae S typhi B subtilis and S epidermidisTherefore theminimum inhibitory concentration (25120583gmL)andminimumbactericidal concentration (50120583gmL) ofNiGswere much lower than the antibiotics

Among fungal pathogens maximum sensitivity wasobserved in C albicans and C tropicalis has showed MICand MFC of NiGs at 25 and 50 120583gmL respectively Mini-mum inhibitory and fungicidal concentration of NiGs werefound to be 50 120583gmL for A clavatus A fumigatus and Aniger MIC and MFC of antibiotic nystatin were observedat 50 and 100 120583gmL for most of the fungal pathogenstested Gram-positive bacteria A clavatus A fumigatus andA niger have showed MIC MBC and MFC of NiGs at50 120583gmL But Gram-negative bacteria C albicans and Ctropicalis have showed higher sensitivity to NiGs at lowerconcentration (25120583gmL) S typhi and C tropicalis recordedhigher MBC and MFC (100 120583gmL) respectively for bothleaf extract and antibiotics Higher antimicrobial activitywas observed in NiGs than antibiotics and leaf extract forall the tested pathogens No significant antibacterial andantifungal activities were observed at NiGs concentrationsless than 25 120583gmL Gram-negative bacteria and Candidaspecies showed relatively higher sensitivity to all testedantimicrobial agents (NiGs antibiotics and leaf extract) thanGram-positive bacteria and Aspergillus species respectivelyIt is clear from the results thatNiGs have enhanced inhibitorybactericidal and fungicidal activities

Higher MIC and MBC values of NiGs in Gram-positive(B subtilis and S epidermidis) pathogens than in Gram-negative bacteria (E coli K pneumonia and S typhi) are


Table 3 Comparison of the antimicrobial activity of NiGs with silver nanoparticles

Organism Antimicrobial effect (MIC) Size (silver nanoparticles) Ref (silver nanoparticles)NiGs (size 12ndash36 nm) (present study) Silver nanoparticles

E coli 25 120583gmL 75 120583gmL 21 nm [2]K pneumoniae 25 120583gmL 11120583gmL 11ndash75 nm [34]S typhi 25 120583gmL 75 120583gmL 21 nm [2]B subtilis 50 120583gmL 70120583gmL 20 nm [35]S epidermidis 50 120583gmL 3125 120583gmL 10ndash50 nm [36]C albicans 25 120583gmL 05mgmL 10ndash30 nm [37]C tropicalis 25 120583gmL 084mgL 25 nm [38]Aspergillus sp 50 120583gmL 40120583gmL 18 nm [39]A fumigatus 50 120583gmL 8120583gmL 20 nm [40]A niger 50 120583gmL 75 120583gmL 20 nm [41]

due to thick peptidoglycan layer in Gram-positive bacteriathat have defended the easy penetration of nanoparticlesthrough the cell membrane Similarly Arokiyaraj et al [17]also reported higher MIC values of green synthesized sil-ver nanoparticles (25 120583gmL) against Gram-positive bacteriathan Gram-negative bacteria (625 120583gmL) Higher MIC andMFC values of NiGs in A clavatusA fumigatus andA nigerthan inC albicansC tropicalis in the present study are due tospore producing and filamentous nature ofAspergillus species(A clavatusA fumigatus andA niger) that decreased fungirsquossensitivity to NiGs This observation is in agreement withprevious reports [13] on antimicrobial properties of biosyn-thesized silver nanoparticles against S aureus S epidermidisE coli A niger and C albicans Enhanced MIC MBCand MFC of NiGs compared to antibiotics and leaf extractare due to nickel nanoparticlersquos larger surface to volumeratio and its penetration to cell membrane This observationconfirmed the earlier findings on antifungal activity [24] andantibacterial activity [25] of silver nanoparticles against Calbicans and of E coli respectively

The antimicrobial activity (MIC) of NiGs was comparedwith the previously reported antimicrobial studies of silvernanoparticles (Table 3) NiGs showed better antimicrobialactivity when compared to silver nanoparticles of similarparticle size Antimicrobial effectiveness of green synthesizedmetal nanoparticles depends on particle dosage treatmenttime and synthesismethods [14]This could be the reason forhigher antimicrobial activity of NiGs than silver nanoparti-cles Variation in antimicrobial activity of antimicrobial activ-ity of NiGs compared to silver nanoparticles of similar sizecould be mainly attributed to the differences in experimentalconditions shape and crystal quality of the nanoparticles asreported in earlier study by Pang et al [26]

34 Growth Curves of Microbial Cells Treated with DifferentConcentrations of NiGs Bacterial growth curves in Figure 2clearly demonstrated the inhibition of bacterial growth atall tested concentrations of NiGs (25ndash100 120583gmL) Culturemedium without NiGs has not shown any inhibition of

growth and also reached stationary phase after 24 hHowevercomplete inhibition was obtained at 50 and 100 120583gmL NiGsfor both Gram-positive (B subtilis and S epidermidis) andGram-negative bacteria (E coli K pneumonia and S typhi)25 120583gmL NiGs could slightly inhibit growth of bacteria butwere not sufficient to outpace the reproduction of bacterialcells From the results it is evidenced that the bactericidalactivity of NiGs increased with increasing NiGs concentra-tion NiGs showed faster inhibition of growth in E coli Kpneumonia and S typhi than B subtilis and S epidermidisMaximum inhibition was observed in E coli at 100 120583gmLNiGs

Inhibitory effects of NiGs on the growth and reproduc-tion of fungal pathogens with respect to concentration areshown in Figure 3 In the absence of NiGs growth of bothtested Candida species (C albicans and C tropicalis) andAspergillus species (A clavatus A fumigatus and A niger)reached exponential phase rapidly However when exposedto 25 120583gmL NiGs growth lagged for longer hours (9 hand 24 h for bacterial and fungal pathogens resp) Furtherincreasing NiGs concentration to 50120583gmL suppressed thefungal growth while 100 120583gmL NiGs completely inhibitedthe growth of fungal pathogens 100 120583gmL NiGs showedmaximum inhibition in C albicans while the effect was lessat lower concentrations (25 50120583gmL) Therefore completegrowth inhibition of bacterial pathogens was at 50120583gmLNiGs while at 100 120583gmL for fungal pathogens

Faster inhibition of growth and reproduction of bacterialand fungal pathogens at 100 120583gmL than 25 and 50 120583gmLNiGs is due to the availability of few nanoparticles and nickelions for inhibition Decreased antimicrobial activity at lowerNiGs concentration is due to the availability of few NiGs forgrowth inhibition as discussed by Sawai [27] for antibacterialactivities of metallic oxide (ZnO MgO and CaO) powdersagainst S aureus and E coli Microbicidal activity of NiGsis also due to the electrostatic interaction between positivelycharged nickel ions and negatively charged microbial cellmembranes These observations are in concurrence withearlier antimicrobial study [16] of zinc oxide nanoparticlesagainst S enterica and C albicans


100120583gmL50120583gmL

25120583gmL0120583gmL

S typhi

50 15 20 2510

Time (h)

00

04

08

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16

OD600

nm

S epidermidis

50 15 20 2510

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E coli

50 15 20 2510

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D600

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OD600

nm

B subtilis

50 15 20 2510

Time (h)

00

04

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16

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OD600

nm

Figure 2 Growth curves of bacterial cells exposed to different concentrations of NiGs

35 NiGs Induced Reactive Oxygen Species (ROS) GenerationReactive oxygen species (ROS) are the natural byproductsof the metabolism [28] Induction of ROS synthesis leadsto the formation of highly reactive radicals that destroy thecells [29] by damaging cell membranes proteins DNA andintracellular system [11] Figures 4(a) and 4(b) showed con-tinuous generation of ROS in bacterial and fungal pathogensat all tested concentrations of NiGs ROS generated byNiGs was concentration-dependent that increased with theincreasingNiGs concentration from 25 to 100120583gmL NiGs at100 120583gmL generated nearly three times higher ROS in E coliK pneumoniae S typhi B subtilis and S epidermidis than atlower concentration (25120583gmL) E coli and K pneumoniaehave showed 81 and 71 ROS generation respectively whileS epidermidis has showed minimal (50) generation of ROSat 100 120583gmL NiGs

Similarly ROS generation at 100 120583gmL NiGs was twotimes higher than in lower concentration (25120583gmL) for alltested fungal pathogens ROS generation was not observed

in both bacterial and fungal control plates without NiGsAmong fungal pathogens treated with NiGs C albicans andC tropicalis showed maximum ROS production while Aniger recorded minimal (32) production of ROSThereforeROS generation was dependent on NiGs concentration andmaximum ROS production was observed in Gram-negativebacteria (65 to 80) and in Candida species (47 to 50)

Increased generation of ROS from bacterial and fungalcells treated with NiGs at all varying concentrations is dueto the reaction of NiGs with water forming ROS (hydro-gen peroxide H

2O2) that damages membrane proteins and

permeability Similar destruction of bacteria due to H2O2

generated by titanium oxide nanoparticles-biofilm interfaceswas earlier discussed byThirunavukkarasu et al (2014) [30]

36 Effect of NiGs on Protein Leak from Microbial CellMembranes Figures 5(a) and 5(b) showed the enhancedleakage of intracellular proteins from microbial membranesinto the extracellular medium at varying concentrations of


100120583gmL50120583gmL

25120583gmL0120583gmL

C albicans

0 2010 40 5030

Time (h)

00

02

04

06

08O

D600

nm

A niger

0 2010 40 5030

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A clavatus

0 2010 40 5030

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nm

Figure 3 Growth curves of fungal cells exposed to different concentrations of NiGs

NiGs (25 50 and 100 120583gmL) No significant protein leakagefrom bacterial and fungal cells was detected in controlProtein leakage from NiGs treated bacterial and fungal cellsincreased with NiGs concentration from 25 to 100120583gmL fora contact period of 6 h and 24 h respectively with maximumleak at 100 120583gmL NiGs

At 100 120583gmL NiGs the amount of protein leakage wasup to 30 27 24 19 and 14 120583gmL (Figure 5(a)) for E coliK pneumoniae S typhi B subtilis and S epidermidisrespectively Protein leakage from E coli K pneumoniae Styphi B subtilis and S epidermidismembranes ranged from19 to 26 15 to 24 14 to 23 10 to 16 and 8 to 12 120583gmLrespectively at 25ndash50120583gmL NiGs The results suggestedthat NiGs could accelerate leakage of protein from bacterialcytoplasm and highest leakage (30 120583gmL) was observed in Ecoli

Similarly elevated protein leakage was observed in alltested fungal pathogens treated with NiGs (Figure 5(b))Protein leakage from C albicans C tropicalis A clavatus

A fumigatus andA niger ranged from 11 to 16 11 to 15 9 to 138 to 13 and 5 to 11120583gmL respectively at 25ndash100120583gmLNiGsC albicans has showed highest protein leakage (16120583gmL)compared to other fungal pathogens

A similar phenomenon of ROS induced oxidative stress-induced leakage of cellular contents from microbial cellmembranes treated with zinc oxide nanoparticles and silvernanoparticles was discussed elsewhere [11 31]

37 Effect of NiGs on Lactate Dehydrogenase (LDH) ActivityThe effect of NiGs on lactate dehydrogenase an importantcytoplasmic enzyme is represented in Figures 6(a) and6(b) Significantly higher leakage of intracellular LDH intoextracellular medium was observed in both bacterial (43ndash95UL) (Figure 6(a)) and fungal (48ndash82UL) (Figure 6(b))cells treated with NiGs than control group LDH leakageinto extracellular medium increased with increase in NiGsconcentration from 25 to 100120583gmL The LDH leakage fromE coli K pneumoniae S typhi B subtilis and S epidermidis


DCF

fluo

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ence

()

S typhiS epidermidis

E coliK pneumoniae

B subtilis

0

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50 100 1500NiGs concentration (120583gmL)

(a)

DCF

fluo

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()


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C albicansA niger

A fumigatusC tropicalis

A clavatus

(b)

Figure 4 Formation of ROS in (a) bacterial and (b) fungal cells exposed to different concentrations of NiGs

Prot

ein

leak

age (120583

gm

L)

0

6

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18

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36



E coliK pneumoniae

B subtilis

(a)

C albicansA niger


A clavatus

0

4

8

12

16

20

Prot

ein

leak

age (120583

gm

L)


(b)

Figure 5 Leakage of proteins from (a) bacterial and (b) fungal cells exposed to different concentrations of NiGs




E coliK pneumoniae

B subtilis

LDH

activ

ity (U

L)

20

40

60

80

100

(a)

LDH

activ

ity (U

L)



E coliK pneumoniae

B subtilis

0

20

40

60

80

(b)

Figure 6 LDH leakage of (a) bacterial and (b) fungal cells exposed to different concentrations of NiGs

in the absence ofNiGs (control) was 55 52 45 36 and 32ULrespectively However the cells treated with 25ndash100120583gmLNiGs showed higher LDH leakage ranging from 69 to 9564 to 89 56 to 83 47 to 79 and 43 to 72UL for E coliK pneumoniae S typhi B subtilis and S epidermidis Themaximum LDH leakage (95UL) was observed in E colicompared to other bacterial pathogens

LDH activity of C albicans C tropicalis A clavatus Afumigatus and A niger at 25ndash100120583gmL NiGs was found tobe 69ndash95 64ndash89 56ndash83 47ndash79 and 43ndash72UL respectivelywhen compared with their respective control (55 52 45 36and 32UL) Increase in LDH leakage with increase in NiGsconcentration suggested that the NiGs enhanced the leakageof intracellular LDH

Enhanced leakage of proteins fromNiGs treated bacterialand fungal cell membranes into culture medium is due togeneration of free radicals from NiGs surface that inducedmembrane damage and leaked membrane and cellular pro-teins as discussed elsewhere [32]

38 Effect of Antioxidant on Microbicidal Activity of NiGsThe involvement of ROS in antimicrobial activity of NiGswas confirmed by using antioxidant ascorbic acid to scav-enge the ROS produced by NiGs The protective activityof 10mM ascorbic acid was observed (Figures 7(a) and7(b)) against microbicidal activity of NiGs against testedpathogens Pathogens treated with 100 120583gmL NiGs (Figures3 and 4) have not showed any growth due to ROS formationHowever in the presence of ascorbic acid both bacterial andfungal pathogens exhibited growth similar to the control

(Figures 3 and 4) Growth curves of bacterial and fungalpathogens in Figures 7(a) and 7(b) confirmed that ascorbicacid was able to protect the cells completely from toxicity ofNiGs It is evidenced from the study that ROSwas involved inthe microbicidal activity of NiGs and ascorbic acid preventedthis antimicrobial activity by scavenging the generated ROSGrowth curves of all tested bacterial and fungal pathogensin the presence of 100 120583gmL NiGs and 10mM ascorbic acidwere similar to the control and this is due to the free radicalscavenging activity of ascorbic acid

39 Mechanism of Antimicrobial Activity of NiGs Figure 8summarizes the interaction of NiGs with microbial cellsThe antimicrobial activity of nickel nanoparticles relies ongeneration of ROS and release of nickel ions Ni (II) Diffusionand endocytosis of NiGs followed by nickel nanoparticlersquosaccumulation in cell membrane altermembrane permeabilityand destroy membrane proteins NiGs react with waterforming ROS that penetrate the cell membrane causing pro-tein disruption and cell membrane damage with subsequentleakage of cellular contents Dissolution of Ni (II) ions andfree radicals interrupt electron transport in the microbialcell resulting in cell death These observations are in goodagreement with earlier reports [19 33] for antimicrobialactivity of silver nanoparticles

4 Conclusions

Green synthesis of nanoparticles providesmore advancementin pharmaceutical and biomedical applications than chemical


0

1

2

S typhi

S epidermidis

E coli

K pneumoniae

B subtilis

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nmO

D600

nm

(a)

0

1

C albicans

A niger

A fumigatus

C tropicalis

A clavatus

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

0

1

OD600

nmO

D600

nm

0

1

2

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

(b)

Figure 7 Effect of ascorbic acid on (a) bactericidal and (b) fungicidal activity of NiGs

and physical methods due to their cost-effectiveness andecofriendliness To overcome the increasing drug resistancepublic health problems and adverse reactions there is anutmost need to develop novel antimicrobial agents Nickelnanoparticles were synthesized from leaf extract of O sanc-tum and characterized as described in our previous study [12]Among tested pathogens Gram-negative bacteria (E coli Kpneumonia and S typhi) and Candida species (C albicansC tropicalis) showed higher growth inhibition and micro-bicidal activity when treated with NiGs than Gram-positivebacteria (B subtilis and S epidermidis) andAspergillus species

(A clavatus A fumigatus and A niger) respectively Enhan-ced antimicrobial activity of NiGs was attributed to the activeformation of ROS that led to the loss of cellular proteinsand LDH through damaged cell membrane resulting in celldeath In conclusion the present study suggested that ROSgenerated from the surface of NiGs interacted and damagedthe cellmembranes resulting in leakage of cellular contents bycell disruption This study reveals the potential of NiGs as anantimicrobial agent at 50120583gmL and hence could be exploitedas antimicrobial coatings on surface of materials for variousenvironmental and biomedical applications


Oxidative damage

Surface bound

Alter membrane permeability

ROS

ROS Perforations

Destroy membrane proteins

Release Ni (II) ions

Diffusion or endocytosis

Membrane rupture

Protein inactivation

Interrupt electron transport

Protein leakage

Intracellular protein

Membrane protein

NiGs

O2

H2O2

minuslowast

O2

minuslowast

eminus

eminus

Figure 8 Mechanism of antimicrobial activity of green synthesized nickel nanoparticles

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] Q A Pankhurst N K T Thanh S K Jones and J DobsonldquoProgress in applications of magnetic nanoparticles in bio-medicinerdquo Journal of Physics D Applied Physics vol 42 no 22pp 1ndash13 2009

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

[3] S Ravikumar R Gokulakrishnan and P Boomi ldquoIn vitroantibacterial activity of the metal oxide nanoparticles againsturinary tract infectious bacterial pathogensrdquo Asian PacificJournal of Tropical Disease vol 2 no 2 pp 85ndash89 2012

[4] D Liu S Ren H Wu Q Zhang and L Wen ldquoMorphologycontrol in synthesis of nickel nanoparticles in the presence ofpolyvinylpyrrolidone (PVPK30)rdquo Journal of Materials Sciencevol 43 no 6 pp 1974ndash1978 2008

[5] A R Joseph and B Viswanathan ldquoSynthesis of nickel nanopar-ticles with fcc and hcp crystal structuresrdquo Indian Journal ofChemistry vol 50 pp 176ndash179 2010

[6] S Dhananasekaran R Palanivel and S Pappu ldquoAdsorptionof methylene blue bromophenol blue and coomassie brilliantblue by 120572-chitin nanoparticlesrdquo Journal of Advanced Researchvol 7 no 1 pp 113ndash124 2016

[7] J Huang Q Li D Sun et al ldquoBiosynthesis of silver and goldnanoparticles by novel sundried Cinnamomum camphora leafrdquoNanotechnology vol 18 no 10 Article ID 105104 pp 105104ndash105115 2007

[8] L-C Chiang L-T Ng P-W Cheng W Chiang and C-C LinldquoAntiviral activities of extracts and selected pure constituentsofOcimum basilicumrdquoClinical and Experimental Pharmacologyand Physiology vol 32 no 10 pp 811ndash816 2005

[9] S Jyoti S Satendra S Sushma T Anjana and S ShashildquoAntistressor activity ofOcimum sanctum (Tulsi) against exper-imentally induced oxidative stress in rabbitsrdquo Methods andFindings in Experimental and Clinical Pharmacology vol 29 no6 pp 411ndash416 2007

[10] C Pellieux A Dewilde C Pierlot and J-M Aubry ldquoBac-tericidal and virucidal activities of singlet oxygen generatedby thermolysis of naphthalene endoperoxidesrdquo Methods inEnzymology vol 319 pp 197ndash207 2000

[11] S-H Kim H-S Lee D-S Ryu S-J Choi and D-S Lee ldquoAnti-bacterial activity of silver-nanoparticles against Staphylococcusaureus and Escherichia colirdquoKorean Journal of Microbiology andBiotechnology vol 39 no 1 pp 77ndash85 2011

[12] C J Pandian R Palanivel and S Dhananasekaran ldquoGreen syn-thesis of nickel nanoparticles using Ocimum sanctum and theirapplication in dye and pollutant adsorptionrdquo Chinese Journal ofChemical Engineering vol 23 no 8 pp 1307ndash1315 2015

[13] S C Yen and M D Mashitah ldquoCharacterization of Agnanoparticles produced by White-Rot Fungi and Its in vitroantimicrobial activitiesrdquo The International Arabic Journal ofAntimicrobial Agents vol 2 no 3 pp 1ndash8 2012


[14] S Gunalan R Sivaraj and V Rajendran ldquoGreen synthesizedZnO nanoparticles against bacterial and fungal pathogensrdquoProgress in Natural Science Materials International vol 22 no6 pp 693ndash700 2012

[15] 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

[16] G Basak D Das and N Das ldquo Dual role of acidic diacetatesophorolipid as biostabilizer for ZnO nanoparticle synthesisand biofunctionalizing agent against Salmonella enterica andCandida albicansrdquo Journal of Microbiology and Biotechnologyvol 24 no 1 pp 87ndash96 2014

[17] S Arokiyaraj M V Arasu S Vincent et al ldquoRapid greensynthesis of silver nanoparticles from Chrysanthemum indicumL and its antibacterial and cytotoxic effects an in vitro studyrdquoInternational Journal of Nanomedicine vol 9 no 1 pp 379ndash3882014

[18] 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

[19] K Mallikarjuna G Narasimha G R Dillip et al ldquoGreensynthesis of silver nanoparticles using Ocimum leaf extract andtheir characterizationrdquo Digest Journal of Nanomaterials andBiostructures vol 6 no 1 pp 181ndash186 2011

[20] R Govindasamy R Jeyaraman J Kadarkaraithangam et alldquoNovel and simple approach using synthesized nickel nanopar-ticles to control blood-sucking parasitesrdquo Veterinary Parasitol-ogy vol 191 pp 332ndash339 2013

[21] X S Wang X Liu L Wen Y Zhou Y Jiang and Z LildquoComparison of basic dye crystal violet removal from aqueoussolution by low-cost biosorbentsrdquo Separation Science and Tech-nology vol 43 no 14 pp 3712ndash3731 2008

[22] A K Suresh D A Pelletier and M J Doktycz ldquoRelatingnanomaterial properties and microbial toxicityrdquoNanoscale vol5 no 2 pp 463ndash474 2013

[23] H F Salem K A M Eid and M A Sharaf ldquoFormulation andevaluation of silver nanoparticles as antibacterial and antifungalagents with a minimal cytotoxic effectrdquo Internation Journal ofDrug Development and Research vol 3 pp 293ndash304 2011

[24] K-J Kim W S Sung B K Suh et al ldquoAntifungal activity andmode of action of silver nano-particles on Candida albicansrdquoBioMetals vol 22 no 2 pp 235ndash242 2009

[25] 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

[26] H Pang F Gao and Q Lu ldquoMorphology effect on antibacterialactivity of cuprous oxiderdquo Chemical Communications vol 7 no9 pp 1076ndash1078 2009

[27] J Sawai ldquoQuantitative evaluation of antibacterial activities ofmetallic oxide powders (ZnO MgO and CaO) by conductimet-ric assayrdquo Journal of Microbiological Methods vol 54 no 2 pp177ndash182 2003

[28] W Lin Y-WHuang X-D Zhou andYMa ldquoToxicity of ceriumoxide nanoparticles in human lung cancer cellsrdquo InternationalJournal of Toxicology vol 25 no 6 pp 451ndash457 2006

[29] J S Kim E Kuk K N Yu et al ldquoAntimicrobial effects ofsilver nanoparticlesrdquo Nanomedicine Nanotechnology Biologyand Medicine vol 3 no 1 pp 95ndash101 2007

[30] S Thirunavukkarasu R Abdul J Chidambaram et al ldquoGreensynthesis of titanium dioxide nanoparticles using Psidiumguajava extract and its antibacterial and antioxidant propertiesrdquoAsian Pacific Journal of Tropical Medicine vol 7 no 12 pp 968ndash976 2014

[31] M Fang J-H Chen X-L Xu P-H Yang andH F HildebrandldquoAntibacterial activities of inorganic agents on six bacteriaassociated with oral infections by two susceptibility testsrdquoInternational Journal of Antimicrobial Agents vol 27 no 6 pp513ndash517 2006

[32] O Choi and Z Hu ldquoSize dependent and reactive oxygen speciesrelated nanosilver toxicity to nitrifying bacteriardquo EnvironmentalScience and Technology vol 42 no 12 pp 4583ndash4588 2008

[33] K Kenneth Y Wong and L Xuelai ldquoSilver nanoparticlesmdashthe real lsquosilver bulletrsquo in clinical medicinerdquoMedicinal ChemistryCommunications vol 1 pp 125ndash131 2010

[34] R K Chrakanth C Ashajyothi A K Oli and C Prabhu-rajeshwar ldquoPotential bactericidal effect of silver nanoparticlessynthesised from Enterococcus speciesrdquo Oriental Journal ofChemistry vol 30 no 3 pp 1253ndash1262 2014

[35] S Agnihotri S Mukherji and S Mukherji ldquoSize-controlledsilver nanoparticles synthesized over the range 5ndash100 nmusing the same protocol and their antibacterial efficacyrdquo RSCAdvances vol 4 no 8 pp 3974ndash3983 2014

[36] Y S Chan andM DMashitah ldquoBiosynthesis of silver nanopar-ticles from Schizophyllum commune and In-vitro antibacterialand antifungal studiesrdquo Journal of Physical Science vol 24 no2 pp 83ndash96 2013

[37] A Nasrollahi Kh Pourshamsian and P Mansourkiaee ldquoAnti-fungal activity of silver nanoparticles on some of fungirdquo Inter-national Journal of Nano Dimension vol 1 no 3 pp 233ndash2392011

[38] A Panacek M Kolar R Vecerova et al ldquoAntifungal activity ofsilver nanoparticles against Candida spprdquo Biomaterials vol 30no 31 pp 6333ndash6340 2009

[39] C Gao Y Xu andC Xu ldquoIn Vitro activity of nano-silver againstocular pathogenic fungirdquo Life Science Journal vol 9 no 4 pp750ndash753 2012

[40] AMonir and SAzar ldquoAntifungal activity of silver nanoparticlesindifferent sizes against some pathogenic fungirdquo Journal ofApplied Chemical Research vol 8 no 4 pp 115ndash122 2014

[41] J Jain S Arora J M Rajwade P Omray S Khandelwal and KM Paknikar ldquoSilver nanoparticles in therapeutics developmentof an antimicrobial gel formulation for topical userdquo MolecularPharmaceutics vol 6 no 5 pp 1388ndash1401 2009

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


synthesized and characterized nickel nanoparticles usingleaf extracts of Ocimum sanctum with 1mM of aqueousnickel nitrate Antimicrobial activity of synthesized nickelnanoparticles was evaluated by various susceptibility assayson bacterial and fungal pathogens The toxicity of NiGs wasstudied against Gram-negative (Escherichia coli Klebsiellapneumoniaeand Salmonella typhi) and Gram-positive (Bacil-lus subtilis Staphylococcus epidermidis) bacterial and fungalpathogens (Candida albicans Candida tropicalis Aspergillusfumigatus Aspergillus clavatusand Aspergillus niger) Wealso substantiate that NiGs induced reactive oxygen speciesformation destroys microbial cell membrane and its perme-ability leading to growth suppression and cell death

2 Materials and Methods

21 Materials and Strains O sanctum leaves were collectedfrom Karaikudi town Tamil Nadu India Taxonomic iden-tification was done by Department of Botany AlagappaUniversity Karaikudi Tamil Nadu India

Lyophilized cultures of Escherichia coli (MTCC 1682)Klebsiella pneumoniae (MTCC 8911) Salmonella typhi(MTCC 3224) Bacillus subtilis (MTCC 6133) Staphylococcusepidermidis (MTCC 7919) Candida albicans (MTCC 3018)Candida tropicalis (MTCC 6222) Aspergillus fumigatus(MTCC 2508) Aspergillus clavatus (MTCC 1323) and Asper-gillus niger (MTCC 281) were procured from Microbial TypeCulture Collection (MTCC) located in Indian Institute ofMicrobial Technology Chandigarh India Mueller-Hintonagar media for bacteria and potato dextrose agar media forfungi were purchased fromHi-Media Laboratories MumbaiIndia Nickel nitrate was purchased from SD Fine ChemicalsLtd Mumbai India All other chemicals were purchasedfrom Sigma-Aldrich Mumbai India

22 Synthesis and Characterization of Nanoparticles Nickelnanoparticles were synthesized using the leaf extract of Osanctum and characterized according to our previous study[12] Chemical interaction of compounds present inOcimumsanctum leaf extract with nanoparticles and morphologyof NiGs were characterized by Fourier transform infrared(FTIR) spectroscopy and transmission electron microscope(TEM) respectively

23 Dose-Dependent Antimicrobial Assay Antibacterial andantifungal assayswere performedwithMueller-Hinton (MH)agar and Sabouraud dextrose (SD) agarmedium respectivelyBacterial and fungal cultures were prepared to 05McFarlandstandards prior to the assay Antimicrobial activity of NiGswas evaluated by disc diffusion assay against the Gram-negative bacteria (E coli K pneumoniae and S typhi)Gram-positive bacteria (B subtilis S epidermidis) and fungi(C albicans C tropicalis A clavatus A fumigatus and Aniger) Pure microbial cultures were subcultured on nutrientagar and uniformly swabbed on individual plates 20120583L ofNiGs at different concentrations (25 50 and 100 120583gmL) wasimpregnated to 6mm filter paper discs dried and placed onthe culture plate Bacterial and fungal cultureswere incubated

at 37∘C for 24 h and 48 h respectively [13] Antimicrobialactivities were studied by the diameter of zone of inhibitionDeionized water (as control) and leaf extract were used tocompare the antimicrobial activity of NiGs

24 Minimum Inhibitory Concentration (MIC) MinimumBactericidal Concentration (MBC) and Minimum Fungici-dal Concentration (MFC) Assay 100 120583L of NiGs in seriallydescending concentrations 200 to 1120583gmL was added tomicrotitre plates with 100120583LMHbroth for bacterial or 100120583LSD broth for fungal assays Dilutions were done by twofoldserial dilution and 100120583L of bacterial and fungal sampleswas inoculated to respective wells Bacterial and fungal plateswere incubated for 24 h and 48 h respectively at 37∘C [13] andoptical densities were determined at 600 nmusingmicroplatereader Antibiotics (amoxicillin (Amx) and nystatin (Nys))were used to compare the bactericidal and fungicidal activityof NiGs

MBC and MFC were determined by subculturing 2 120583Lof above MIC serial dilution after 24 h of incubation inrespective wells containing 100120583L of broth per well Bacterialand fungal colonies were quantified by further incubationfor 24 h and 48 h respectively at 37∘C MBC and MFC werethe lowest concentration of nanoparticles or antibiotics thatprevented the growth of bacterial and fungal colonies on solidmedia respectively [14]

25 Determination of Microbial Growth Kinetics in thePresence of NiGs Microbial growth rate was observed byinoculating the microtitre plates with MH and SD brothcontaining 105 colony forming units (cfu) per mL of bacterialor fungal pathogens respectively and loaded with varyingconcentrations of nanoparticles (0 25 50 and 100 120583gmL)The plates were incubated at 37∘C and shaken at 180 rpmAfter inoculation the optical density (OD) at 600 nm [15]was serially monitored at every 3 h interval till 24 h andevery 6 h interval till 48 h for bacterial and fungal pathogensrespectively

26 Detection of Reactive Oxygen Species (ROS) Reactiveoxygen species produced in microbial cells were determinedby 2101584071015840 dichlorofluorescein diacetate (DCFDA) Intracellularesterase cleaves fluorescence based probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) to polar impermeable non-fluorescent molecule that accumulates intracellularly and itssubsequent oxidation yields highly fluorescent product 2101584071015840dichlorofluorescein (DCF) which was monitored by increasein fluorescence 105 cfumL bacterial and fungal pathogenswere treated with 0 25 50 and 100 120583gmL NiGs and incu-bated at 37∘C for 6 h and 24 h respectively After incubationthe cultures were centrifuged at 5000 rpm for 10min at 4∘CThe supernatant was treated with 100 120583M DCFDA for 1 hand the ROS formed (fluorescence intensity of DCF) wasdetermined at 488 nm excitation wavelength and 535 nmemission wavelength using fluorescence spectrophotometer[11]

























12

4

3

5

SEBS

3

2

14

5

3KP

4

2

1

5

EC

2

5

1

3

45

2

31

4

ST


(a)



12

4

3

5

CA

1

5

2

4

3

CT3

5

4

21

AF

1 23

4

5

ANAC

3

2

1

5

4


(b)


























100120583gmL50120583gmL

25120583gmL0120583gmL

S typhi

50 15 20 2510

Time (h)

00

04

08

12

16

OD600

nm

S epidermidis

50 15 20 2510

Time (h)

00

04

08

12

16

20

OD600

nm

E coli

50 15 20 2510

Time (h)

00

04

08

12O

D600

nmK pneumoniae

50 15 20 2510

Time (h)

00

04

08

12

16

OD600

nm

B subtilis

50 15 20 2510

Time (h)

00

04

08

12

16

20

OD600

nm











100120583gmL50120583gmL

25120583gmL0120583gmL

C albicans

0 2010 40 5030

Time (h)

00

02

04

06

08O

D600

nm

A niger

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm

A fumigatus

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nmC tropicalis

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nm

A clavatus

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm









DCF

fluo

resc

ence

()


E coliK pneumoniae

B subtilis

0

20

40

60

80

100


(a)

DCF

fluo

resc

ence

()


0

10

20

30

40

50

60

C albicansA niger


A clavatus

(b)


Prot

ein

leak

age (120583

gm

L)

0

6

12

18

24

30

36



E coliK pneumoniae

B subtilis

(a)

C albicansA niger


A clavatus

0

4

8

12

16

20

Prot

ein

leak

age (120583

gm

L)


(b)





E coliK pneumoniae

B subtilis

LDH

activ

ity (U

L)

20

40

60

80

100

(a)

LDH

activ

ity (U

L)



E coliK pneumoniae

B subtilis

0

20

40

60

80

(b)








4 Conclusions



0

1

2

S typhi

S epidermidis

E coli

K pneumoniae

B subtilis

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nmO

D600

nm

(a)

0

1

C albicans

A niger

A fumigatus

C tropicalis

A clavatus

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

0

1

OD600

nmO

D600

nm

0

1

2

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

(b)





Oxidative damage

Surface bound


ROS

ROS Perforations




Membrane rupture



Protein leakage


Membrane protein

NiGs

O2

H2O2

minuslowast

O2

minuslowast

eminus

eminus




References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



























12

4

3

5

SEBS

3

2

14

5

3KP

4

2

1

5

EC

2

5

1

3

45

2

31

4

ST


(a)



12

4

3

5

CA

1

5

2

4

3

CT3

5

4

21

AF

1 23

4

5

ANAC

3

2

1

5

4


(b)


























100120583gmL50120583gmL

25120583gmL0120583gmL

S typhi

50 15 20 2510

Time (h)

00

04

08

12

16

OD600

nm

S epidermidis

50 15 20 2510

Time (h)

00

04

08

12

16

20

OD600

nm

E coli

50 15 20 2510

Time (h)

00

04

08

12O

D600

nmK pneumoniae

50 15 20 2510

Time (h)

00

04

08

12

16

OD600

nm

B subtilis

50 15 20 2510

Time (h)

00

04

08

12

16

20

OD600

nm











100120583gmL50120583gmL

25120583gmL0120583gmL

C albicans

0 2010 40 5030

Time (h)

00

02

04

06

08O

D600

nm

A niger

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm

A fumigatus

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nmC tropicalis

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nm

A clavatus

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm









DCF

fluo

resc

ence

()


E coliK pneumoniae

B subtilis

0

20

40

60

80

100


(a)

DCF

fluo

resc

ence

()


0

10

20

30

40

50

60

C albicansA niger


A clavatus

(b)


Prot

ein

leak

age (120583

gm

L)

0

6

12

18

24

30

36



E coliK pneumoniae

B subtilis

(a)

C albicansA niger


A clavatus

0

4

8

12

16

20

Prot

ein

leak

age (120583

gm

L)


(b)





E coliK pneumoniae

B subtilis

LDH

activ

ity (U

L)

20

40

60

80

100

(a)

LDH

activ

ity (U

L)



E coliK pneumoniae

B subtilis

0

20

40

60

80

(b)








4 Conclusions



0

1

2

S typhi

S epidermidis

E coli

K pneumoniae

B subtilis

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nmO

D600

nm

(a)

0

1

C albicans

A niger

A fumigatus

C tropicalis

A clavatus

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

0

1

OD600

nmO

D600

nm

0

1

2

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

(b)





Oxidative damage

Surface bound


ROS

ROS Perforations




Membrane rupture



Protein leakage


Membrane protein

NiGs

O2

H2O2

minuslowast

O2

minuslowast

eminus

eminus




References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






12

4

3

5

SEBS

3

2

14

5

3KP

4

2

1

5

EC

2

5

1

3

45

2

31

4

ST


(a)



12

4

3

5

CA

1

5

2

4

3

CT3

5

4

21

AF

1 23

4

5

ANAC

3

2

1

5

4


(b)


























100120583gmL50120583gmL

25120583gmL0120583gmL

S typhi

50 15 20 2510

Time (h)

00

04

08

12

16

OD600

nm

S epidermidis

50 15 20 2510

Time (h)

00

04

08

12

16

20

OD600

nm

E coli

50 15 20 2510

Time (h)

00

04

08

12O

D600

nmK pneumoniae

50 15 20 2510

Time (h)

00

04

08

12

16

OD600

nm

B subtilis

50 15 20 2510

Time (h)

00

04

08

12

16

20

OD600

nm











100120583gmL50120583gmL

25120583gmL0120583gmL

C albicans

0 2010 40 5030

Time (h)

00

02

04

06

08O

D600

nm

A niger

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm

A fumigatus

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nmC tropicalis

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nm

A clavatus

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm









DCF

fluo

resc

ence

()


E coliK pneumoniae

B subtilis

0

20

40

60

80

100


(a)

DCF

fluo

resc

ence

()


0

10

20

30

40

50

60

C albicansA niger


A clavatus

(b)


Prot

ein

leak

age (120583

gm

L)

0

6

12

18

24

30

36



E coliK pneumoniae

B subtilis

(a)

C albicansA niger


A clavatus

0

4

8

12

16

20

Prot

ein

leak

age (120583

gm

L)


(b)





E coliK pneumoniae

B subtilis

LDH

activ

ity (U

L)

20

40

60

80

100

(a)

LDH

activ

ity (U

L)



E coliK pneumoniae

B subtilis

0

20

40

60

80

(b)








4 Conclusions



0

1

2

S typhi

S epidermidis

E coli

K pneumoniae

B subtilis

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nmO

D600

nm

(a)

0

1

C albicans

A niger

A fumigatus

C tropicalis

A clavatus

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

0

1

OD600

nmO

D600

nm

0

1

2

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

(b)





Oxidative damage

Surface bound


ROS

ROS Perforations




Membrane rupture



Protein leakage


Membrane protein

NiGs

O2

H2O2

minuslowast

O2

minuslowast

eminus

eminus




References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



























100120583gmL50120583gmL

25120583gmL0120583gmL

S typhi

50 15 20 2510

Time (h)

00

04

08

12

16

OD600

nm

S epidermidis

50 15 20 2510

Time (h)

00

04

08

12

16

20

OD600

nm

E coli

50 15 20 2510

Time (h)

00

04

08

12O

D600

nmK pneumoniae

50 15 20 2510

Time (h)

00

04

08

12

16

OD600

nm

B subtilis

50 15 20 2510

Time (h)

00

04

08

12

16

20

OD600

nm











100120583gmL50120583gmL

25120583gmL0120583gmL

C albicans

0 2010 40 5030

Time (h)

00

02

04

06

08O

D600

nm

A niger

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm

A fumigatus

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nmC tropicalis

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nm

A clavatus

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm









DCF

fluo

resc

ence

()


E coliK pneumoniae

B subtilis

0

20

40

60

80

100


(a)

DCF

fluo

resc

ence

()


0

10

20

30

40

50

60

C albicansA niger


A clavatus

(b)


Prot

ein

leak

age (120583

gm

L)

0

6

12

18

24

30

36



E coliK pneumoniae

B subtilis

(a)

C albicansA niger


A clavatus

0

4

8

12

16

20

Prot

ein

leak

age (120583

gm

L)


(b)





E coliK pneumoniae

B subtilis

LDH

activ

ity (U

L)

20

40

60

80

100

(a)

LDH

activ

ity (U

L)



E coliK pneumoniae

B subtilis

0

20

40

60

80

(b)








4 Conclusions



0

1

2

S typhi

S epidermidis

E coli

K pneumoniae

B subtilis

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nmO

D600

nm

(a)

0

1

C albicans

A niger

A fumigatus

C tropicalis

A clavatus

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

0

1

OD600

nmO

D600

nm

0

1

2

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

(b)





Oxidative damage

Surface bound


ROS

ROS Perforations




Membrane rupture



Protein leakage


Membrane protein

NiGs

O2

H2O2

minuslowast

O2

minuslowast

eminus

eminus




References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials














100120583gmL50120583gmL

25120583gmL0120583gmL

S typhi

50 15 20 2510

Time (h)

00

04

08

12

16

OD600

nm

S epidermidis

50 15 20 2510

Time (h)

00

04

08

12

16

20

OD600

nm

E coli

50 15 20 2510

Time (h)

00

04

08

12O

D600

nmK pneumoniae

50 15 20 2510

Time (h)

00

04

08

12

16

OD600

nm

B subtilis

50 15 20 2510

Time (h)

00

04

08

12

16

20

OD600

nm











100120583gmL50120583gmL

25120583gmL0120583gmL

C albicans

0 2010 40 5030

Time (h)

00

02

04

06

08O

D600

nm

A niger

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm

A fumigatus

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nmC tropicalis

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nm

A clavatus

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm









DCF

fluo

resc

ence

()


E coliK pneumoniae

B subtilis

0

20

40

60

80

100


(a)

DCF

fluo

resc

ence

()


0

10

20

30

40

50

60

C albicansA niger


A clavatus

(b)


Prot

ein

leak

age (120583

gm

L)

0

6

12

18

24

30

36



E coliK pneumoniae

B subtilis

(a)

C albicansA niger


A clavatus

0

4

8

12

16

20

Prot

ein

leak

age (120583

gm

L)


(b)





E coliK pneumoniae

B subtilis

LDH

activ

ity (U

L)

20

40

60

80

100

(a)

LDH

activ

ity (U

L)



E coliK pneumoniae

B subtilis

0

20

40

60

80

(b)








4 Conclusions



0

1

2

S typhi

S epidermidis

E coli

K pneumoniae

B subtilis

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nmO

D600

nm

(a)

0

1

C albicans

A niger

A fumigatus

C tropicalis

A clavatus

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

0

1

OD600

nmO

D600

nm

0

1

2

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

(b)





Oxidative damage

Surface bound


ROS

ROS Perforations




Membrane rupture



Protein leakage


Membrane protein

NiGs

O2

H2O2

minuslowast

O2

minuslowast

eminus

eminus




References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




100120583gmL50120583gmL

25120583gmL0120583gmL

S typhi

50 15 20 2510

Time (h)

00

04

08

12

16

OD600

nm

S epidermidis

50 15 20 2510

Time (h)

00

04

08

12

16

20

OD600

nm

E coli

50 15 20 2510

Time (h)

00

04

08

12O

D600

nmK pneumoniae

50 15 20 2510

Time (h)

00

04

08

12

16

OD600

nm

B subtilis

50 15 20 2510

Time (h)

00

04

08

12

16

20

OD600

nm











100120583gmL50120583gmL

25120583gmL0120583gmL

C albicans

0 2010 40 5030

Time (h)

00

02

04

06

08O

D600

nm

A niger

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm

A fumigatus

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nmC tropicalis

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nm

A clavatus

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm









DCF

fluo

resc

ence

()


E coliK pneumoniae

B subtilis

0

20

40

60

80

100


(a)

DCF

fluo

resc

ence

()


0

10

20

30

40

50

60

C albicansA niger


A clavatus

(b)


Prot

ein

leak

age (120583

gm

L)

0

6

12

18

24

30

36



E coliK pneumoniae

B subtilis

(a)

C albicansA niger


A clavatus

0

4

8

12

16

20

Prot

ein

leak

age (120583

gm

L)


(b)





E coliK pneumoniae

B subtilis

LDH

activ

ity (U

L)

20

40

60

80

100

(a)

LDH

activ

ity (U

L)



E coliK pneumoniae

B subtilis

0

20

40

60

80

(b)








4 Conclusions



0

1

2

S typhi

S epidermidis

E coli

K pneumoniae

B subtilis

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nmO

D600

nm

(a)

0

1

C albicans

A niger

A fumigatus

C tropicalis

A clavatus

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

0

1

OD600

nmO

D600

nm

0

1

2

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

(b)





Oxidative damage

Surface bound


ROS

ROS Perforations




Membrane rupture



Protein leakage


Membrane protein

NiGs

O2

H2O2

minuslowast

O2

minuslowast

eminus

eminus




References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




100120583gmL50120583gmL

25120583gmL0120583gmL

C albicans

0 2010 40 5030

Time (h)

00

02

04

06

08O

D600

nm

A niger

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm

A fumigatus

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nmC tropicalis

0 2010 40 5030

Time (h)

00

04

08

12

16

OD600

nm

A clavatus

0 2010 40 5030

Time (h)

00

04

08

12

16

20

OD600

nm









DCF

fluo

resc

ence

()


E coliK pneumoniae

B subtilis

0

20

40

60

80

100


(a)

DCF

fluo

resc

ence

()


0

10

20

30

40

50

60

C albicansA niger


A clavatus

(b)


Prot

ein

leak

age (120583

gm

L)

0

6

12

18

24

30

36



E coliK pneumoniae

B subtilis

(a)

C albicansA niger


A clavatus

0

4

8

12

16

20

Prot

ein

leak

age (120583

gm

L)


(b)





E coliK pneumoniae

B subtilis

LDH

activ

ity (U

L)

20

40

60

80

100

(a)

LDH

activ

ity (U

L)



E coliK pneumoniae

B subtilis

0

20

40

60

80

(b)








4 Conclusions



0

1

2

S typhi

S epidermidis

E coli

K pneumoniae

B subtilis

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nmO

D600

nm

(a)

0

1

C albicans

A niger

A fumigatus

C tropicalis

A clavatus

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

0

1

OD600

nmO

D600

nm

0

1

2

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

(b)





Oxidative damage

Surface bound


ROS

ROS Perforations




Membrane rupture



Protein leakage


Membrane protein

NiGs

O2

H2O2

minuslowast

O2

minuslowast

eminus

eminus




References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




DCF

fluo

resc

ence

()


E coliK pneumoniae

B subtilis

0

20

40

60

80

100


(a)

DCF

fluo

resc

ence

()


0

10

20

30

40

50

60

C albicansA niger


A clavatus

(b)


Prot

ein

leak

age (120583

gm

L)

0

6

12

18

24

30

36



E coliK pneumoniae

B subtilis

(a)

C albicansA niger


A clavatus

0

4

8

12

16

20

Prot

ein

leak

age (120583

gm

L)


(b)





E coliK pneumoniae

B subtilis

LDH

activ

ity (U

L)

20

40

60

80

100

(a)

LDH

activ

ity (U

L)



E coliK pneumoniae

B subtilis

0

20

40

60

80

(b)








4 Conclusions



0

1

2

S typhi

S epidermidis

E coli

K pneumoniae

B subtilis

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nmO

D600

nm

(a)

0

1

C albicans

A niger

A fumigatus

C tropicalis

A clavatus

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

0

1

OD600

nmO

D600

nm

0

1

2

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

(b)





Oxidative damage

Surface bound


ROS

ROS Perforations




Membrane rupture



Protein leakage


Membrane protein

NiGs

O2

H2O2

minuslowast

O2

minuslowast

eminus

eminus




References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






E coliK pneumoniae

B subtilis

LDH

activ

ity (U

L)

20

40

60

80

100

(a)

LDH

activ

ity (U

L)



E coliK pneumoniae

B subtilis

0

20

40

60

80

(b)








4 Conclusions



0

1

2

S typhi

S epidermidis

E coli

K pneumoniae

B subtilis

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nmO

D600

nm

(a)

0

1

C albicans

A niger

A fumigatus

C tropicalis

A clavatus

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

0

1

OD600

nmO

D600

nm

0

1

2

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

(b)





Oxidative damage

Surface bound


ROS

ROS Perforations




Membrane rupture



Protein leakage


Membrane protein

NiGs

O2

H2O2

minuslowast

O2

minuslowast

eminus

eminus




References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

1

2

S typhi

S epidermidis

E coli

K pneumoniae

B subtilis

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0 2010 30

Time (h)

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

0

1

OD600

nmO

D600

nm

(a)

0

1

C albicans

A niger

A fumigatus

C tropicalis

A clavatus

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

Time (h)0 20 6040

0

1

OD600

nmO

D600

nm

0

1

2

OD600

nm

0

1

OD600

nm

0

1

OD600

nm

(b)





Oxidative damage

Surface bound


ROS

ROS Perforations




Membrane rupture



Protein leakage


Membrane protein

NiGs

O2

H2O2

minuslowast

O2

minuslowast

eminus

eminus




References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Oxidative damage

Surface bound


ROS

ROS Perforations




Membrane rupture



Protein leakage


Membrane protein

NiGs

O2

H2O2

minuslowast

O2

minuslowast

eminus

eminus




References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










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Documents

Research Article Screening Antimicrobial Activity of …downloads.hindawi.com/journals/jnp/2016/4694367.pdfResearch Article Screening Antimicrobial Activity of Nickel Nanoparticles