Size-dependent antimicrobial response of zinc oxide nanoparticles

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Published in IET NanobiotechnologyReceived on 9th April 2012Revised on 13th September 2012Accepted on 19th September 2012doi: 10.1049/iet-nbt.2012.0008

T Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 111–117oi: 10.1049/iet-nbt.2012.0008

ISSN 1751-8741

Size-dependent antimicrobial response of zinc oxidenanoparticlesLoganathan Palanikumar1, Sinna Nadar Ramasamy1, Chandrasekaran Balachandran2

1Crystal Growth Centre, Anna University, Chennai 600025, India2Division of Microbiology, Entomology Research Institute, Loyola College, Chennai 600034, India

E-mail: [email protected]

Abstract: Antibacterial and antifungal activities of zinc oxide nanoparticles (ZnO NPs) were investigated against infectiousmicroorganisms. ZnO NPs were prepared by wet chemical precipitation method varying the pH values. Particle size andmorphology of the as-prepared ZnO powders were characterised by X-ray diffraction, Fourier transform infrared spectroscopyand transmission electron microscope. The zone of inhibition by NPs ranged from 0 to 17 mm. The lowest minimuminhibitory concentration value of NPs is 25 µg.ml−1 against Staphylococcus epidermidis. These studies demonstrate that ZnONPs have wide range of antimicrobial activities towards various microorganisms. The results obtained in the authors’ studyindicate that the inhibitory efficacy of ZnO NPs is significantly dependent on its chosen concentration and size. Significantinhibition in antibacterial response was observed for S. epidermidis when compared with control antibiotic.

1 Introduction

As particles are reduced from a micrometre to nanometer size,the resulting properties can change dramatically. For example,electrical conductivity, hardness, active surface area,chemical reactivity and biological activity are all known tobe altered. Medicinal sciences are investigating the use ofnanotechnology to improve medical diagnosis andtreatments [1–3]. The bactericidal effectiveness of metalNPs has been suggested to be because of both particle sizeand high surface-to-volume ratio. Such characteristicsshould allow them to interact closely with bacterialmembranes, rather than the effect being solely because ofthe release of metal ions [4]. Inorganic antibacterial agentsare more stable at high temperatures and pressurescompared with the organic materials, and the metallic oxidepowders could be suggested as powerful antimicrobialagents [5].Zinc oxide is traditionally used for the photocatalytic

oxidation of organic and inorganic pollutants and sensitisersfor the photodestruction of cancer cells, bacteria and virusesvia oxidative damage [6–8]. NPs generally produces toxiceffects with plasma proteins, transient inflammatory, cellinjury effects leading to inflammation and fibrosis. The NPshave shown elevated blood biochemical parameters,accumulation of foamy alveolar macrophages, degeneratedalveolar macrophages indicating alveolar lipoproteinosis. Inhumans, some types of NPs which are used for cosmeticsetc. may result in skin disease. However, ZnO#ZnSquantum dots heterojunctions have been reported toenhance transportation of flavonoid glycosides in blood[7, 8]. Hence, it is appropriate to assume that ZnO isnon-toxic and so it has been taken for this research study.

ZnO NPs are widely used in many consumer products likecosmetics, toothpaste, textiles and skin lotions [9].Staphylococcus aureus and S. epidermidis are naturalinhabitants of human and animal skin, but it can sometimescause infections that affect many organs [10]. Thepathogenic bacteria Klebsiella pneumoniae andEnterobacter aerogenes are the major causative agents ofnosocomial infections [11]. Since 1980s,methicillin-resistant S. aureus (MRSA) has been commonlylinked with hospital-associated infections [12]. Paratyphoidfever is caused by Salmonella enterica serotypes ParatyphiA, Paratyphi B or Paratyphi C. As per literature, anestimated 5 400 000 cases of paratyphoid fever occurredglobally in 2000 [13]. Serotype Paratyphi B var. L (+)tartrate (+) causes a typical Salmonella gastroenteritisinstead of enteric fever [14]. Candida albicans andMalassezia pachydermatis are the most frequent human andanimal pathogens [15, 16]. The antibacterial and antifungalactivities of bulk ZnO powders and ZnO NPs have beendemonstrated already [12, 17]. However, little is knownabout the activity of ZnO NPs in the range 15–50 nm rangetowards the infectious microorgansims. Towards thispurpose, evaluation has been made on the antibacterial andantifungal effect as a function of synthesised ZnO NPs sizebased on the inhibition zone in the disk diffusion tests withdetermining minimum inhibitory concentration (MIC).

2 Materials and methods

2.1 Synthesis of ZnO NPs

ZnO NPs were synthesised with a slight modificationsuggested by Wu et al. [18] from aqueous solutions of zinc

111& The Institution of Engineering and Technology 2014

Fig. 1 XRD pattern of ZnO NPs prepared at different pH

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nitrate [Zn (NO3)2.6H2O; purchased from Fischer Chemicals,Mumbai, India; purity 96%] and hexamethyltetramine (HMT;C6H12N4; purchased from Qualigens chemicals Ltd.,Mumbai, India; purity 99%). The two chemicals weremixed separately with milli-Q water to a concentration of0.05 M for the [Zn (NO3)2] solution, and 1.5 M for theHMT solution. The separate solutions are stirred for 30 mineach, and then mixed with 130 rpm stirring. The solutionswas adjusted to the desired pH (5.0, 6.0 and 7.2) andheated to 80°C for 45 min. The product is collected bycentrifugation (Compufuge, Remi Electrotechnik Limited,Thane, India). The ammonium hydroxide solution (1 N)was added to pH 5.0 synthesised solutions to enhance theformation of ZnO at 80°C [19].
2.2 Particle characterisation

Powder X-ray diffraction (XRD, Seifert, JSO-DE BYEFLEX2002, Germany) was utilised to identify the crystalline phasecomposition and purity. The phase was found to be hexagonaland no impurity peaks were found. The morphology and grainsize of the ZnO were observed by TECNAI G2 Model T-30S-twin high-resolution transmission electron microscopy(HRTEM). The quality of the ZnO NPs was analysed byFourier transform infrared spectroscopy (FTIR, PerkinElmer Spectrum One).

2.3 Microbial organisms

The following bacteria and fungi were used for the experiment.Bacteria: Salmonella paratyphi B,K. pneumoniaeMTCC 109,Bacillus subtilis MTCC 441, E. aerogenes MTCC 111,Staphylococcus epidermidis MTCC 3615, Methicillinresistant-MRSA. The reference cultures were obtained fromInstitute of Microbial Technology (IMTECH; Chandigarh,India 160 036); fungi: C. albicans MTCC 227 andM. pachydermatis were obtained from the Department ofMicrobiology (Christian Medical College, Vellore, TamilNadu, India).

2.4 Antimicrobial assay

Antibacterial and antifungal activities were carried out usingdisc-diffusion method [20, 21]. Petri plates were preparedwith 20 ml of sterile Mueller Hinton agar (MHA) (Himedia,Mumbai). The test cultures were swabbed on the top of thesolidified media and allowed to dry at room temperature for10 min. The suspension of NPs (in milli-Q water) wassonicated to prepare required suspensions such as 50,100 and 200 µg.ml−1 added to each well (diameter of 8mm) separately. Preliminarily, a broad range ofconcentrations (10–200 µg.ml−1) were chosen forantimicrobial assay (data not shown). Based on thoseresults, three concentrations were chosen for the presentwork as a function of size. The suspension of NPs wereloaded on the wells of the medium and left for 30 min atroom temperature for diffusion. Negative control wasprepared using respective solvents. Streptomycin (25 µg/disc; purchased from Himedia Chemicals Ltd., Mumbai,India) was used as positive control against bacteria.Ketoconazole (25 µg/disc; purchased from HimediaChemicals Ltd., Mumbai, India) was used as positivecontrol for fungi. The plates were incubated for 24 h at 37°Cfor bacteria and for 48 h at 28°C for fungi. Zones ofinhibition were recorded in millimetres by repeating theexperiment thrice for each concentration.


2.5 Minimum inhibitory concentration

MIC studies of the NPs were performed according to thestandard reference methods for bacteria [22], forfilamentous fungi [21] and yeasts [23]. The requiredconcentrations (3.125, 6.25, 12.5, 25, 50, 100 and 200 µg.ml−1) of the NPs were dispersed in milli-Q water anddiluted to give serial two-fold dilutions that were added toeach medium in 96 well plates. A volume of 100 µl(inoculum) from each well was inoculated. The antifungalagents ketoconazole for fungi and streptomycin for bacteriawere included in the assays as positive controls. For fungi,the plates were incubated for 48–72 h at 28°C and forbacteria the plates were incubated for 24 h at 37°C. TheMIC for fungi was defined as the lowest extractconcentration, showing no visible fungal growth afterincubation time. 5 µl of tested broth was placed on thesterile MHA plates for bacteria and incubated at respectivetemperatures. The MIC for bacteria was determined as thelowest concentration of the compound inhibiting the visualgrowth of the test cultures on the agar plate.

2.6 Statistical analysis

The differences in antimicrobial activity of ZnO NPs incomparison with control were assessed by one-way analysisof variance [24]. Dunnett’s post hoc test was employed tocompare the significant difference between control anddifferent exposure concentrations. This statistical analysiswas carried out using computer-assisted software programGraph Pad Prism version 5.0. Other statistical analysis wascarried out using Microsoft Office Excel 2003.

3 Results

The present study employed a low-temperature synthesismethod to prepare ZnO NPs. Zinc nitrate and HMTsolutions were mixed at 80°C for 45 min at different pHand a precipitation reaction occurred, after the pH was 8.2.The XRD pattern of synthesised ZnO NPs demonstratedthat the ZnO is crystalline in nature, and the diffractionpeaks matched very well with a hexagonal zincite (wurtzite)phase of ZnO (Fig. 1). The diffraction pattern andinter-planar spacing closely matched those in the standarddiffraction pattern of ZnO (Powder diffraction file ICDD

IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 111–117doi: 10.1049/iet-nbt.2012.0008

Fig. 3 FTIR spectrum of as-prepared ZnO NPs (38 nm)

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36-1451 a = 3.249 Å and c = 5.206 Å). The XRD peaks show(100), (002), (101), (102), (110), (103), (200) and (201)reflection lines of hexagonal zincite phase of ZnO particles.No characteristic peaks of any impurities were detectedsuggesting good quality and widened peak were detected.The particle size based on broadening was analysed byScherrer formula, modified form of Williamson–Hallanalysis and size–strain plot method. The crystalline sizecan be calculated using the following equation
dhklbhklcosu( )2= K/D d2hklbhklcosu

( )+ (1/2)2

where K is the constant that depends on the shape of the particles.The particle size determined from the slope of linearly fitted dataand the root of the y-intercept gives the strain [25].The quality of the as-synthesised product without any heat

treatment was analysed by the FTIR spectroscopy. FTIRconfirm the formation of ZnO. Fig. 2 shows the FTIRspectrum acquired in the range of 400–4000 cm−1. Theband at 535 cm−1 corresponds to the stretching vibration ofZn–O bond. The broadening corresponding to Zn–O peakat 535 cm−1 is broadened. This may be because of thenanocrystalline nature of the compound. The broadabsorption bands in the range 3900–2350 and 1637 cm−1

correspond to the presence of the surface hydroxyl groups[26]. C–OH stretching (1387 cm−1) are detected from theFTIR spectrum [27]. The band at 1387 cm−1 corresponds tothe CH2 deformation and absorption bands at 1235–1125cm−1 are responsible for CN stretch. The band at 998 and897 cm−1 corresponds to CH2 rock [28]. Although theseextra peaks because of the starting materialhexamethylenetetraamine appear in the as-prepared material,the tetramethyl amine is expected to go-off on heattreatment, before we go for antimicrobial and MIC studies.The morphology and size distribution of the ZnO NPs

prepared at various pH are shown in Fig. 3. It can beclearly observed that the size of NPs was reduced with

Fig. 2 TEM images of ZnO NPs prepared at different pH

a As preparedb pH6.0c pH5.0


decreasing pH. The number of hexagonal-shaped NPsincreased as the pH decreased. The TEM images confirmedthe formation of hexagonal structure of ZnO and are inagreement with the XRD results. The particle size of theZnO nanograins prepared at different pH was 38 ± 2; 25 ± 4and 15 ± 4 nm in as-prepared (pH 7.2), pH 6.0 and pH 5.0,respectively. Modified Scherrer formula gives 32 ± 4; 25 ± 4and 15 ± 4 nm, respectively, for the above samples.

3.1 Antimicrobial and MIC studies

The antibacterial and antifungal activity of ZnO NPs wascompared for infectious Gram-positive and Gram-negativebacteria and fungus. The results of antimicrobial activity ofdifferent size of ZnO NPs were shown in Table 1 andFigs. 4a–h. The zone of inhibition (in mm) reflects themagnitude of susceptibility of the microorganism. Thestrains susceptible to NPs exhibit larger zone of inhibition,whereas resistant strains exhibit smaller or no zone ofinhibition. Significant difference between control antibiotics(streptomycin and ketoconazole) were observed for all theNPs treatment, whereas insignificant zone of inhibition wereobserved for S. epidermis. No antimicrobial response wasobserved for 25 and 38 nm ZnO NPs against K. pneumonia.Table 2 presents the MICs of ZnO NPs for infectiousmicroorganisms. Highest MIC response was observed forS. epidermis.

4 Discussion

The re-emergence of infectious diseases poses a serious threatto public health worldwide, and the increasing rate of theappearance of antibiotic-resistant strains in a short period oftime within both Gram-positive and Gram-negative bacteriaand fungal microorganisms is a major public health concern[12]. Alternative therapeutics to control and prevent thespread of infections in both community and hospitalenvironments are required [29].ZnO NPs are much more effective agents in controlling

the growth of various microorganisms [12, 30, 31]. Ourpreliminary studies show that the reduced particle sizehad greater efficacy in inhibiting the growth ofmicroorganisms. Similarly, metals and metal oxides suchas ZnO NPs are known to be toxic to host human and


Table 1 Antimicrobial activity of ZnO NPs (zone of inhibition in mm; 200 µg.ml−1)

Organism 15 ± 4 nm ZnO NPs 25 ± 4 nm ZnO NPs 38 ± 2 nm ZnO NPs Control

Gram negativeB. subtilis 12.00 ± 1.00a 5.67 ± 0.58a 5.33 ± 0.58a 22 ± 0.84S. paratyphi B 7.67 ± 0.58a 6.33 ± 0.58a 5.67 ± 0.58a 18 ± 0.71K. pneumoniae 5.67 ± 0.58a - - 20 ± 0.71

Gram positiveS. epidermidis 17.00 ± 1.00b 15.67 ± 0.58b 13.47 ± 0.58b 14 ± 0.71E. aerogenes 6.00 ± 1.00a 5.67 ± 0.58a 6.00 ± 1.00a 22 ± 0.71S. aureus MRSA 6.67 ± 0.58a 6.00 ± 1.00a 5.33 ± 0.58a 30 ± 0.90

fungiC. albicans 5.67 ± 0.58a 6.00 ± 1.00a 5.33 ± 0.58a 28 ± 0.71M. pachydermatis 5.00 ± 1.00a 6.00 ± 0.45a 5.00 ± 0.25a 26 ± 0.90

(-) no activityaStatistically significant P < 0.05; P < 0.01 when compared to controlbStatistically insignificant when compared to control antibiotic (streptomycin – antibacterial; ketoconazole – antifungal)

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plant cells at relatively higher concentration and lowerparticle size [12, 32]. They are not expected to be toxic atvery low concentrations. In fact, it has been shown thatZnO protects against intestinal diseases by protectingintestinal cells from Escherichia coli infection byinhibiting the adhesion and internalisation of bacteria [33].Therefore antibacterial activity of ZnO NPs from thepresent results suggests that ZnO NPs may playdifferential response to various tested microorganisms.This may be consistent with the prediction thatS. epidermis can metabolise Zn2+ as an oligoelement [33].Similarly, metal-ion homaeostasis is important for bacteriallife because of their involvement in the regulation of awide array of metabolic functions as coenzymes, cofactorsand catalysts, and as structural stabilisers of enzymes andDNA-binding proteins [34]. However, excess metal ormetal ions are toxic for bacterial cells. Therefore certainbacteria have developed mechanisms to regulate the influxand efflux processes to maintain the steady intracellularconcentration of metal ions, including the Zn2+ ion. Zincis essential for all organisms, because it plays a criticalrole in the catalytic activity and/or structural stability ofmany proteins [35]. Limited studies have reported themechanisms and regulation of zinc transport in bacteria[35, 36]. Several studies suggest that bacteria appear topossess a specific energy-dependent zinc transport system[37].The genes responsible for the transport of zinc ions have

been characterised in several bacteria, including S. aureusand B. subtilis [12]. In S. aureus, ZntA and ZntR geneshave been characterised, and it has been shown that ZntA, atransmembrane protein, is responsible for the efflux of zincand cobalt ions and that ZntR encodes for a Zn-responsiveregulatory protein [12, 35]. In Salmonella typhimurium,ZnuABC genes encode a high-affinity zinc uptake systemand pitA gene is responsible for low-affinity system [38].Studies have reported that ATP-binding protein ZurA isresponsible for zinc uptake in S. epidermis (source:UniprotKB/TrEMBL; accession no: E6JMH7). Metal NPsbreaks down the membrane permeability barrier and it maybe possible that ZnO NPs perturbs the membrane lipidbilayer in case of fungal organisms [39].Much of the differential antibacterial activity of ZnO NPs

on various microorganisms will depend on cell wallintegrity or membrane structures of the respective bacteriaas the outer membrane structure of Gram-negative


bacteria is remarkably different from that of Gram-positivebacteria. It is also well-known that different strains withina species vary significantly in terms of infectivity, andtolerance to various agents including antibiotics [28, 40].The higher antibacterial activity of ZnO NPs inS. epidermis may involve the production of reactiveoxygen species and the deposition on the surface oraccumulation in the cytoplasm of the cells as observed inearlier studies for S. aureus [40]. However, no inhibitionis observed for K. pneumoniae, which requires furtherinvestigations. The highest concentration (200 µg.ml−1)and lowest particle size 15 nm has been found to bestrongly inhibit the survival of pathogenic microorganismstested. The results obtained in our study indicate that theinhibitory efficacy of ZnO NPs is very much dependenton its chosen concentration and size, which is similar toother findings [6, 32, 41].These primary findings suggest that ZnO NP not exceeding

25 nm can be used externally to control the spreading ofbacterial infections. In the prevention and control ofbacterial spreading and infections, the main target is the cellwall structure. The cell wall of most pathogenic bacteria iscomposed of surface proteins for adhesion and colonisation,and components such as polysaccharides and teichoic acidthat protect against host defenses and environmentalconditions [41]. These components are chargedmacromolecules; therefore specific interactions to disrupttheir main function and the cell membrane location may betriggered by introducing specific groups on the surface ofthe NPs. It has been reported that certain long-chainpolycations coated onto surfaces can efficiently kill bothGram-positive and Gram-negative bacteria [42, 43]. Thesestudies have indicated that families of unrelatedhydrophobic groups are equally efficient in killing bacteria[12]. The present study suggests that ZnO NPs might haveinhibited the growth of fungi by interfering with cellfunction and causing deformation in fungal hyphae [31].Several studies such as electron microscopic imaging andRaman spectroscopy have reported the antifungal andantibacterial effects of ZnO NPs and their interaction bytraditional microbiological plating [12, 31]. Althoughpossible mechanisms have been proposed in earlier reports[12, 41], still the exact mechanism underlying theantimicrobial activity of the ZnO NPs remains to beunderstood. It can be concluded that ZnO NPs constitute aneffective antimicrobial agent against common pathogenic


Fig. 4 Antimicrobial activity of ZnO NPs against

a B. subtilis MTCC 441b S. paratyphi Bc K. pneumoniae MTCC 109d S. epidermidis MTCC 3615e E. aerogenes MTCC 111f S. aureus MRSAg C. albicans TCC 227h M. pachydermatisControl – streptomycin (antibacterial) and ketoconazole (antifungal)

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Table 2 MIC of ZnO NPs

Organism 15 ± 4 nmZnO NPs

25 ± 4 nmZnO NPs

38 ± 2 nmZnO NPs

Control

Gram negativeB. subtilis 50 200 >200 <0.78S. paratyphi B 100 100 200 6.25K. pneumoniae 200 >200 >200 6.25S. typhimurium >200 >200 >200 >100

Gram positiveS. epidermidis 25 25 50 6.25E. aerogenes 100 200 200 <0.78S. aureus MRSA 200 200 200 <0.78

FungiC. albicans 200 200 200 25M. pachydermatis 200 200 200 12.5

Control: streptomycin – antibacterial; ketoconazole – antifungal

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microorganisms. Further study and research are needed to findout the exact mechanism of membrane damage of microbialcells caused due to ZnO NPs.

5 Acknowledgment

Professor Dr. S. Ramasamy, CSIR Emeritus Scientist andDr. L. Palanikumar, CSIR-ResearchAssociate sincerelyacknowledge the receipt of CSIR Emeritus Scientist Scheme,New Delhi, India (21(1074)/08/EMR–II dated 28.04.2008).The authors thank National Centre for Nanoscience andTechnology, University of Madras, Chennai for providingHRTEM facilities.

6 References

1 Bennet, D.J., Schuurbiers, D.: ‘Nanobiotechnology: responsibleaction on issue in society and ethics’, NSTI-Nanotech., 2005, 2,pp. 765–768

2 Howard, K.A., Kjems, J.: ‘Polycation-based nanoparticle delivery forimproved RNAi therapeutics’, Expert Opin. Biol. Ther., 2007, 7,pp. 1811–1822

3 Andersen, S., Dong, M., Nielsen, M.M., et al.: ‘Self-assembly of anano-scale DNA box with a controllable lid’, Nature, 2009, 459,pp. 73–76

4 Morones, J.R., Elechiguerra, J.L., Camacho, A., Holt, K., Kouri, J.B.,Ramirez, J.T.: ‘The bactericidal effect of silver nanoparticles’,Nanotechnology, 2005, 16, pp. 2346–2353

5 Sawai, J.: ‘Quantitative evaluation of antibacterial activities of metallicoxide powders (ZnO, MgO and CaO) by conductimetric assay’,J. Microbiol. Methods, 2003, 54, pp. 177–182

6 Feris, K., Otto, C., Tinker, J., et al.: ‘Electrostatic interactions affectnanoparticle-mediated toxicity to gram-negative bacteriumPseudomonas aeruginosa PAO1’, Langmuir, 2010, 26, pp. 4429–4436

7 Xiao, J., Zhao, Y., Mao, F., Wu, M., Yu, X.: ‘Investigation the toxiceffect of QDs heterojunction on the interaction between smallmolecules and plasma proteins by fluorescence and resonancelight-scattering spectra’, Analyst, 2012, 137, pp. 195–201

8 Xiao, J., Kai, G., Chen, X.: ‘Effect of CdTe QDs on the protein-druginteraction’, Nanotoxicology, 2012, 6, pp. 304–314

9 Mortimer, M., Kasemets, K., Kahru, A.: ‘Toxicity of ZnO and CuOnanoparticles to ciliated protozoa Tetrahymena thermophile’,Toxicology, 2010, 269, pp. 182–189

10 Bhunia, A.K. (Ed.): ‘Foodborne microbial pathogens: mechanisms andpathogenesis’ (Springer LLC, New York, USA, 2008)

11 Saonuam, P., Hiransuthikul, N., Suankratay, C., Malathum, K.,Dancha-ivijitr, S.: ‘Risk factors for nosocomial infections caused byextended spectrum β-lactamase producing Escherichia coli or Klebsiellapneumonia in Thailand’, Asian Biomed., 2008, 2, pp. 485–491

12 Jones, N., Ray, B., Ranjit, K.T., Manna, A.C.: ‘Antibacterial activity ofZnO nanoparticle suspensions on a broad spectrum of microorganisms’,FEMS Microbiol. Lett., 2008, 279, pp. 71–76


13 Crump, J.A., Luby, S.P., Mintz, E.D.: ‘The global burden of typhoidfever’, Bull. WHO, 2004, 82, pp. 346–353

14 Prager, R., Rabsch, W., Streckel, W., Voigt, W., Tietze, E., Tschape, H.:‘Molecular properties of Salmonella enterica serotype paratyphi Bdistinguish between its systemic and its enteric pathovars’, J. Clin.Microbiol., 2003, 41, pp. 4270–4278

15 Marcon, M.J., Powell, D.A.: ‘Human infections due toMalassezia spp.’,Clin. Microbiol. Rev., 1992, 5, pp. 101–119

16 Sanglard, D., Odds, F.C.: ‘Resistance of Candida species to antifungalagents: molecular mechanisms and clinical consequences’, LancetInfect. Dis., 2002, 2, pp. 73–85

17 Sawai, J., Yoshikawa, T.: ‘Quantitative evaluation of antifungal activityof metallic oxide powders (MgO, CaO and ZnO) by an indirectconductimetric assay’, J. Appl. Microbiol., 2004, 96, pp. 803–809

18 Wu, P.F., Pike, J., Zhang, F., Chan, S.W.: ‘Low-temperature synthesis ofzinc oxide nanoparticles’, Int. J. Appl. Ceram. Technol., 2006, 3,pp. 272–278

19 Palanikumar, L., Ramasamy, S., Hariharan, G., Balachandran, C.:‘Influence of particle size of nano zincoxide on the controlled deliveryof amoxicillin’, Appl. Nanosci., 2012, pp. 1–11

20 Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., Yolke, R.H.:‘Manual of clinical microbiology’ (ASM, Washington DC, 1995)

21 Clinical and Laboratory Standards Institute (CLSI) 2008. Referencemethod for Broth dilution antifungal susceptibility testing offilamentous fungi; approved standard second edition. CLSI documentM38-A2 (ISBN 1-56238-668-9). Clinical and Laboratory StandardsInstitute, 940, West valley Road, Suite 1400, Wayne, Pennsylvania19087-1898, USA

22 Duraipandiyan, V., Ignacimuthu, S.: ‘Antibacterial and antifungalactivity of Flindersine isolated from the traditional medicinal plant,Toddalia asiatica (L.) Lam’, J. Ethnopharmacol., 2009, 123,pp. 494–498

23 NCCLS 2002. M27-A2. In National Committee for Clinical LaboratoryStandards. Reference method for broth dilution antifungal susceptibilitytesting of yeasts: proposed standard

24 Zar, J.H.: ‘Biostatistical analysis’ (Prentice-Hall International Editions,USA, 1996), pp. 662

25 Zak, A.K., Abd Majid, W.H., Abrishami, M.E., Yousefi, R.: ‘X-rayanalysis of ZnO nanoparticles by Williamson-Hall and size–strain plotmethods’, Solid-State Sci., 2011, 13, pp. 251–256

26 Sharma, P.K., Pandey, A.C., Zolnierkiewicz, G., Guskos, N., Rudowicz,C.: ‘Relationship between oxygen defects and the photoluminescenceproperty of ZnO nanoparticles: a spectroscopic view’, J. Appl. Phys.,2009, 106, pp. 094314–1

27 Song, R., Liu, Y., He, L.: ‘Synthesis and characterization ofmercaptoacetic acid-modified ZnO nanoparticles’, Solid-State Sci.,2008, 10, pp. 1563–1567

28 Bernstein, M.P., Sandford, S.A., Allamandola, L.J., Chang, S.:‘Infrared spectrum of matrix-isolated hexamethylenetetramine in Arand H20 at cryogenic temperatures’, J. Phys. Chem., 1994, 98, pp.12206–12210

29 Wang, L., Barrett, J.F.: ‘Control and prevention of MRSA infections’,Methods Mol. Biol., 2007, 391, pp. 209–225

30 Kasemets, K., Ivask, A., Dubourguier, H.C., Kahru, A.: ‘Toxicity ofnanoparticles ZnO, CuO and TiO2 to yeast Saccharomycescerevisiae’, Toxicol. In Vitro, 2009, 23, pp. 1116–1122

31 He, L., Liu, Y., Mustapha, A., Lin, M.: ‘Antifungal activity of zinc oxidenanoparticles against Botrytis and Penicillium expansum’, Microbiol.Res., 2011, 166, pp. 207–215

32 Yin, H., Casey, P.S., McCall, M., Fenech, M.: ‘Effects of surfacechemistry on cytotoxicity, genotoxicity, and the generation of reactiveoxygen species induced by ZnO nanoparticles’, Langmuir, 2010, 26,pp. 15399–15408

33 Roselli, M., Finamore, A., Garaguso, I., Britti, M.S., Mengheri, E.: ‘Zincoxide protects cultured enterocytes from the damage induced by E. coli’,J. Nutr., 2003, 133, pp. 4077–4082

34 Padmavathy, N., Vijayaragaghavan, R.: ‘Enhanced bioactivity of ZnOnanoparticles – an antimicrobial study’, Sci. Technol. Adv. Mater.,2008, 9, pp. 035004

35 Gaballa, A., Wang, T., Ye, R.W., Helmann, J.D.: ‘Functional analysis ofthe Bacillus subtilis Zur regul-ion’, J. Bacteriol., 2002, 184,pp. 6508–6514

36 Lee, J.W., Helmann, J.D.: ‘Functional specialization within the Furfamily of metalloregulators’, Biometals, 2007, 20, pp. 485–499

37 Lindsay, J.A., Foster, S.J.: ‘Zur: aZn 21-responsive regulatoryelement of Staphylococcus aureus’, Microbiology, 2001, 147, pp.1259–1266

38 Campoy, S., Jara, M., Busquets, N., Perez de Rozas, A.M., Badiola, I.,Barbe, J.: ‘Role of the high-affinity zinc uptake ZnuABC system in


www.ietdl.org
Salmonella enterica serovar Typhimurium virulence’, Infect Immun.,2002, 70, pp. 4721–4725
39 Kim, K.J., Sung, W.S., Suh, B.K., et al.: ‘Antifungal activity and modeof action of silver nano-particles on Candida albicans’, Biometals, 2009,22, pp. 235–242

40 Raghupathi, K.R., Koodali, R.T., Manna, A.C.: ‘Size-dependentbacterial growth inhibition and mechanism of antibacterial activity ofzinc oxide nanoparticles’, Langmuir, 2011, 27, pp. 4020–4028


41 Wahab, R., Kim, Y.S., Mishra, A., Yun, S.I., Shin, H.S.: ‘Formationof ZnO micro-flowers prepared via solution process andtheir antibacterial activity’, Nanoscale Res. Lett., 2010, 5, pp. 1675–1681

42 Tiller, J.C., Liao, C.J., Lewis, K., Klibanov, A.M.: ‘Designing surfacesthat kill bacteria on contact’, Proc. Natl. Acad. Sci. USA, 2001, 98,pp. 5981–5985

43 Berry, V., Gole, A., Kundu, S., Murphy, C.J., Saraf, R.F.: ‘Deposition ofCTAB-terminated nanorods on bacteria to form highly conductinghybrid systems’, J. Am. Chem. Soc., 2005, 127, pp. 17600–17601


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Size-dependent antimicrobial response of zinc oxide nanoparticles