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Antibacterial activity and mechanism of Ag-
ZnO nanocomposite on S. aureus and GFP-
expressing antibiotic resistant E-coli
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Colloids and Surfaces B: Biointerfaces 115 (2014) 359– 367
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces
jou rn al hom epage: www.elsevier.com/locate/colsurfb
Antibacterial activity and mechanism of Ag–ZnO nanocomposite on
S.
aureus and GFP-expressing antibiotic resistant E. coli
Ishita Matai, Abhay Sachdev, Poornima Dubey, S. Uday Kumar,
Bharat Bhushan, P. Gopinath∗
Nanobiotechnology Laboratory, Centre for Nanotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
a r t i c l e i n f o
Article history:
Received
27 April 2013
Received
in revised form
25
November 2013
Accepted
2 December 2013
Available online 18 December 2013
Keywords:Multi-resistant organisms
NanocompositesBactericidalNanoparticlesAntibacterial mechanisma b s t r a c t
Emergence of multi-resistant organisms (MROs) leads to ineffective treatment with the currently avail-
able medications which pose a great threat to public health and food technology sectors. In this regard,
there is an urgent need to strengthen the present therapies or to look over for other potential alternatives
like use of “metal nanocomposites”. Thus, the present study focuses on synthesis of silver–zinc oxide
(Ag–ZnO) nanocomposites which will have a broad-spectrum antibacterial activity against Gram-positive
and Gram-negative bacteria. Ag–ZnO nanocomposites of varied molar ratios were synthesized by sim-
ple microwave assisted reactions in the absence of surfactants. The crystalline behavior, composition
and morphological analysis of the prepared powders were evaluated by X-ray diffraction, infrared
spectroscopy, field emission scanning electron microscopy (FE-SEM) and atomic absorption spectropho-
tometry (AAS). Particle size measurements were carried out by transmission electron microscopy (TEM).
Staphylococcus aureus and recombinant green fluorescent protein (GFP) expressing antibiotic resistant
Escherichia coli were selected as Gram-positive and Gram-negative model systems respectively and the
bactericidal activity of Ag–ZnO nanocomposite was studied. The minimum inhibitory concentration (MIC)
and minimum killing concentration (MKC) of the nanocomposite against the model systems were deter-
mined by visual turbidity analysis and optical density analysis. Qualitative and quantitative assessments
of its antibacterial effects were performed by fluorescent microscopy, fluorescent spectroscopy and Gram
staining measurements. Changes in cellular morphology were examined by atomic force microscopy
(AFM), FE-SEM and TEM. Finally, on the basis of the present investigation and previously published
reports, a plausible antibacterial mechanism of Ag–ZnO nanocomposites was proposed.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
With the increase in emergence and re-emergence of multidrug
resistant pathogens especially antibiotic resistant bacterial strains,
fungi and parasites has become a serious problem to health care and
food technology sectors [1]. Microbes acquire resistance to various
drugs to shield themselves against all odds and develop favorable
modifications to enable its comfortable survival and multiplica-
tion under extreme conditions. Safety concerns associated with
the drug resistant microbes and continuing emphasis on health
care costs have stressed on the need for modifications in the tra-
ditional antimicrobial compounds or search for other promising
alternatives [2].
In the recent era, nanotechnology has blossomed as an
extremely powerful and versatile nano weapon to combat this
∗Corresponding author. Tel.: +91 1332 285650; fax: +91 1332 273560.
E-mail addresses: pgopifnt@iitr.ernet.in , genegopi@gmail.com ,
nanobiogopi@gmail.com (P. Gopinath).particular issue concerning pathogen cessation. Microbes find
difficult to acquire resistance toward nanoparticles as they target
multiple bacterial components, contrary to the mechanistic action
of antibiotics. With regard to nanomaterials, nano-hybrid crystals
with distinct morphologies especially metal–oxide nanocompos-
ites have drawn attention as they combine the properties of the
constituent elements to exert a more pronounced and synergistic
effect [3].
Amidst various metal nanoparticles, silver has always been the
most interesting and promising candidate to be investigated for its
inhibitory and antibacterial properties since ancient times [4,5] .
However, silver is associated with the problem of aggregation upon
reduction in its size which considerably affects its chemical and
antibacterial properties. To overcome this problem, silver can be
capped with polymers like chitosan to form polymeric nanocom-
posites [6] or capped with a layer of metal–oxide, such as zinc
oxide, magnesium oxide and calcium oxide exhibiting core–shell
morphology that offers large surface area to volume ratio.
Recently, zinc oxide has been popularized among the five zinc
compounds as it was recognized as a safe material by the U.S Food
0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2013.12.005

360 I. Matai et al. / Colloids and Surfaces B: Biointerfaces 115 (2014) 359 – 367
and Drug Administration [(21CFR182.8991) (FDA, 2011)] and its
antibacterial activity has been long known [7]. Hence, Ag–ZnO
nanocomposites offer potential applications in food packaging
industry for development of antimicrobial coatings and for coating
surgical instruments to prevent nosocomial infections.
Till date, several methods have been reported for synthe-
sis of Ag–ZnO nanocomposites, such as sol–gel method [8],
co-precipitation method, hydrothermal method [9] pulsed laser
deposition [10], plasma-assisted chemical vapor deposition [11]
and thermolysis [12]. However, all of these methodologies employ
extreme temperature and pressure conditions and long reaction
times which limit their usage on commercial and laboratory scale.
In the present work, Ag–ZnO nanocomposites have been suc-
cessfully synthesized by a simple and rapid method using a
domestic microwave oven and simple metal precursors without
the need for any surfactants. The formulated heterostructure com-
bines the antibacterial properties of both Ag and ZnO particles
at nanoscale to emerge as a broad-spectrum antibacterial agent.
Expression of GFP in Escherichia coli enables easy and rapid qual-
itative and quantitative assessment of the bactericidal efficacy of
the as-synthesized Ag–ZnO nanocomposites.
2. Materials and methods
2.1. Chemicals, bacterial strains and growth media
Ag–ZnO nanocomposites of varied molar ratios were syn-
thesized by simple microwave assisted reactions using 1200 W
domestic microwave oven (IFB), with 2.45 × 109Hz working fre-
quency. Precursors, zinc nitrate (Zn(NO 3)2·6H2O), silver nitrate
and citric acid were purchased from Himedia, Merck Pvt. Ltd.
and S.D. Fine-Chem. Ltd. (India), respectively. Recombinant GFP
expressing E. coli was prepared as mentioned in [13] and S. aureus
(MTCC 737) was obtained from IMTECH, India. Bacterial growth
medium, such as Luria-Bertani (LB) medium and nutrient broth
(NB) medium, were purchased from Merck (Germany) and Hime-
dia (India), respectively. Ampicillin antibiotic was procured from
Sisco research laboratories (India). All the chemicals were of ana-
lytical grade and used as received. All the preparations were made
in Milli-Q water.
2.2. Synthesis of Ag–ZnO nanocomposites
In a typical procedure, zinc nitrate (0.1 M) and silver nitrate with
different molar ratios Zn:Ag (1:0.006, 1:0.06, 1:0.3, 1:0.88) were
added to 8% aqueous citric acid solution and magnetically stirred
to obtain a transparent solution. Then the transparent solution was
heated at 80◦C for 30 min to form a gel. The gel was then subjected
to microwave treatment for 5–25 cycles (each cycle consists of 30 s
on and 30 s off mode to prevent excessive boiling of the solvent).
Obtained brownish fluffy solids were then washed with absolute
ethanol and dried at 100◦C for 20 min. Finally, the resultant powder
was calcined at 500◦C for 2 h in a muffle furnace to obtain dark
brown colored nanopowders as the final product.
2.3. Antibacterial activity of Ag–ZnO nanocomposites
2.3.1. Determination of MIC and MKC of Ag–ZnO nanocomposite
Ag–ZnO nanocomposite with Zn:Ag (1:0.88) molar ratio was
evaluated for its broad-spectrum antibacterial activity against S.
aureus and GFP expressing antibiotic resistant E. coli. Minimum
inhibitory concentration (MIC) and minimum killing concentra-
tion (MKC) of the nanocomposite were determined by growing GFP
E. coli cells in LB medium comprising different concentrations of the
Ag–ZnO nanocomposite (200 /H9262g, 300 /H9262g, 400 /H9262g, 500 /H9262g, 550 /H9262g,600 /H9262g/mL) and S. aureus in NB medium containing various concen-
trations of the Ag–ZnO nanocomposite (20 /H9262g, 30 /H9262g, 40 /H9262g, 50 /H9262g,
60 /H9262g, 70 /H9262g/mL). The bacterial cultures were incubated overnight
at 37◦C at 225 rpm. Growth of the cultures was determined by
visual observation. Culture tube with lowest concentration of the
nanocomposite which exhibited no growth was taken as the MIC
value and the tubes lacking turbidity were re-inoculated in fresh
medium to obtain the MKC value. The minimal concentration of the
nanocomposite that prevented the growth of bacterial cells upon
re-inoculation was considered as its MKC value. In a separate exper-
iment, mixture of Ag and ZnO nanopowders equal in weight to the
obtained MIC and MKC values of the nanocomposite were added to
GFP E. coli bacteria and were left for overnight incubation. Growth
was monitored by visual turbidity analysis upon re-inoculation in
fresh LB medium.
2.3.2. Optical density and fluorescence spectroscopic analysis
Antibacterial activity of the Ag–ZnO nanocomposite [Zn:Ag
(1:0.88)] against S. aureus and GFP expressing E. coli model sys-
tems was investigated by optical density analysis of the treated
and untreated bacteria (control) at 600 nm using UV-Visible spec-
trophotometer (Lasany double-beam L1 2800) and GFP-associated
fluorescence using a Hitachi F-4600 fluorescence spectrophotome-
ter (/NAKexcitation = 410 nm).
2.4. Characterization of Ag–ZnO nanocomposites
Purity and crystalline phase structure of the as-synthesized
Ag–ZnO nanocomposites was recorded using a Bruker AXS
D8 advance powder X-ray diffractometer (Cu-K /H9251radiation,
/NAK = 1.5406 ˚A) in the range of 20–80◦at a scan speed of 0.5◦/min.
Fourier transform infrared spectra (FTIR) of the nanocomposites
were recorded using a Thermo Nicolet FTIR spectrometer in the
range 4000–400 cm−1using KBr pellets. TEM (FEI TECHNAI G2)
operating at 200 keV and FE-SEM (FEI Quanta 200F) equipped
with energy dispersive X-ray detector (EDX) operating at an
accelerating voltage of 15–20 keV were used to determine the par-
ticle size, morphology and compositional analysis of the Ag–ZnO
nanocomposites. Wet-chemical analysis of the nanocomposite was
performed using AAS (Avanta M, GBC Scientific Equipment) with
pure Ag (2, 3, 4 /H9262g/mL) and pure Zn (0.5, 1, 1.5 /H9262g/mL) standards
as reference. The interaction of Ag–ZnO nanocomposites with S.
aureus and GFP E. coli was observed using TEM by placing 10–20 /H9262L
of the samples on carbon-coated copper TEM grids followed by
air-drying the sample.
2.5. Fluorescence and optical microscopic analysis
For fluorescence microscopic analysis, 5 /H9262L of the treated and
untreated GFP E. coli bacterial samples were placed over the micro-
scopic slides to form a thin smear and were then viewed after
air-drying using a Nikon Eclipse LV100 microscope attached with
a B-2A filter at an excitation wavelength of 445–495 nm. Similarly,
5 /H9262L of the treated and untreated S. aureus bacterial samples were
heat fixed and imaged after staining with Gram stain.
2.6. Atomic force microscopic analysis
To study the fine topological changes on the bacterial mem-
brane due to the bactericidal activity of Ag–ZnO nanocomposites,
15–20 /H9262L of treated and untreated S. aureus and GFP E. coli samples
were placed on glass cover slip and air-dried. AFM imaging was per-
formed using an AFM (NTEGRA PNL) operated in a tapping mode
with Si cantilevers having a spring constant of 21 N/m operating at

I. Matai et al. / Colloids and Surfaces B: Biointerfaces 115 (2014) 359 – 367 361
Fig. 1. (a) Typical XRD patterns of as-synthesized Ag–ZnO nanocomposites with
varied molar ratios. (b) FTIR spectra of Ag–ZnO nanocomposites [Zn:Ag (1:0.88)].
a resonance frequency of 160 kHz. The images were acquired at a
scan field of 5 /H9262m × 5 /H9262m.
2.7. Plasmid DNA isolation
Plasmid DNA containing GFP and ampicillin resistant gene
was isolated from the untreated and Ag–ZnO nanocomposite
(550 /H9262g/mL and 400 /H9262g/mL) treated E. coli cells by alkaline lysis
method and analyzed by agarose gel electrophoresis.
2.8. Release of ions by AAS analysis
The release of Ag and Zn ions from Ag–ZnO nanocomposite upon
contact with the water was studied. MIC and MKC concentrations
(550 and 600 /H9262g/mL) of Ag–ZnO were incubated with water at 37◦C
and 225 rpm for different time intervals. Subsequently, the samples
were subjected to AAS analysis for the presence of ions.
3. Results and discussion
3.1. Characterization of Ag–ZnO nanocomposites
3.1.1. XRD analysis
Crystalline behavior and structural properties of Ag–ZnO
nanocomposites were revealed by powder X-ray diffraction
measurements. XRD patterns of the as-synthesized Ag–ZnO
nanocomposites with different molar ratios are shown in Fig. 1a.
The obtained diffraction patterns were analyzed by PANalytical
X’Pert High Score Plus. From the obtained XRD patterns the lat-
tice parameters were calculated and are shown in Table S1. For
Ag–ZnO molar ratios (1:0.006, 1:0.06, 1:0.3) it was observed that
with the increase in the Ag content the intensity of diffraction peakscorresponding to face centered cubic structure of Ag (marked with
‘*’) increased and a slight shift of ZnO peaks to lower 2/DC2 angles
was observed compared to pure ZnO with hexagonal wurtzite
structure (JCPDS card# 36-1451, a = 3.25 ˚A and c = 5.21 ˚A). This
may be attributed to the substitution of some Ag+ions (ionic
radius = 0.126 nm) for Zn2+(ionic radius = 0.074 nm) in the ZnO lat-
tice. The variation in the lattice parameters ‘a’ and ‘c’ is clearly
evident in Table S1. However, the XRD peaks for Zn–Ag (1:0.88)
indicate the formation of clear, distinct phases for both Ag and
ZnO. In this particular molar ratio the slight shift of ZnO peaks to
higher degree is due to shrinkage of the lattice parameters ‘a’ and
‘c’ from 3.25 ˚A to 3.20 ˚A and from 5.21 ˚A to 5.16 ˚A. However, negli-
gible variation in the lattice parameter for cubic Ag was observed
and the 2/DC2 values obtained were in agreement with the pure Ag
(JCPDS card# 04-0783, a = 4.09 ˚A). Formation of separate phases for
both Ag and ZnO in the Zn–Ag (1:0.88) molar ratio is indicative of
the nanocomposite formation. Additionally, the intense diffraction
peak corresponding to reflections (1 1 1) signifies the preferred ori-
entation of growth of the crystallite. No other peaks were identified
suggesting purity of the synthesized samples.
3.1.2. FTIR measurements
The typical IR spectrum of Ag–ZnO nanocomposite [Zn:Ag
(1:0.88)] is given in Fig. 1b. The presence of broad band at
3436.30 cm−1corresponds to the stretching vibration of the O H
mode [14]. This may be due to the hydroxyl groups of water on
ZnO surface covering the surface of Ag. Peak at 1639.13 cm−1is
attributed to the O H bending mode due to adsorption of water
molecules in the sample either during mixing or formation of KBr
pellets [14]. The presence of additional bands at 1400, 2921.74,
2369.57 cm−1corresponds to stretching vibration of N O bond
in nitrate groups [15], symmetrical and asymmetrical stretch-
ing vibrations of C H in CH3and CH2groups of citric acid,
respectively. Peaks at 1091.30, 804.35 and 708.7 cm−1are assigned
to Zn O Zn, Zn O H and Zn O Zn stretching frequencies and
bending frequencies, respectively. Occurrence of significant band
at 477.75 cm−1is characteristic of the formation of Zn O bond [16].
Peak at 652.17 cm−1can be attributed to the Ag–ZnO nanocompos-
ite formation [17].
3.1.3. Morphological, size and compositional estimations of
Ag–ZnO nanocomposite
The shape features, surface morphology and size determination
of the as-synthesized Ag–ZnO nanocomposites [Zn:Ag (1:0.88)]
were investigated using TEM and FE-SEM and results are shown in
Fig. 2(a–e). Fig. 2a represents TEM image of Ag–ZnO nanocomposite
[Zn:Ag (1:0.88)], inset shows the corresponding SAED pattern, (b
and c) are HR-TEM images of Ag–ZnO nanocomposite at different
magnifications, inset in (c) depicts the corresponding EDX pattern.
Formation of nanocomposites was clear from the TEM micrograph
obtained. In Fig. 2(b and c) red arrows represent the less dense ZnO
capping over the more dense Ag nanoparticles (yellow arrows).
The HR-TEM images clearly depict the density differences in
both ZnO and Ag. The obtained EDX confirms the covering of less
dense/porous ZnO layer over the comparatively denser Ag nanopar-
ticles. Silver nanoparticles with average particle size 53.07 nm
were capped with a layer of zinc oxide with thickness around
10.9 nm. Fig. 2d exhibits a typical high-magnification FE-SEM
image depicting formation of uniform spherical nanoparticles. The
weight percentage of silver and zinc present in the nanocomposites
was estimated by EDX analysis (Fig. 2e). An increment in the weight
% of silver from 1.06 to 48.65 and alteration in weight % of zinc
from 92.06 to 48.03 was observed when the Zn:Ag molar ratio was
varied from 1:0.006 to 1:0.88. From the EDX analysis, the elemental
wt% information obtained indicates that Ag–ZnO nanocomposites
prepared using Zn:Ag molar ratio 1:0.88 comprised nearly equal

362 I. Matai et al. / Colloids and Surfaces B: Biointerfaces 115 (2014) 359 – 367
Fig. 2. (a) TEM image of Ag–ZnO nanocomposite [Zn:Ag (1:0.88)], inset shows the corresponding SAED pattern, (b and c) HR-TEM images of Ag–ZnO nanocomposite at different
magnifications,
inset in (c) depicts the corresponding EDX pattern. High magnification FE-SEM image of Ag–ZnO nanocomposite [Zn:Ag (1:0.88)] (d) and its corresponding
EDX pattern (e).
weight % of Ag and Zn, when compared to the other nanocom-
posites (Fig. 2e). In addition, Ag–ZnO nanocomposite (2 ppm or
2 /H9262g/mL) was subjected to AAS analysis with pure Ag and Zn to find
out the unknown concentration of Ag and Zn in the synthesized
Ag–ZnO nanocomposite. Based on the AAS findings, the concen-
trations of Ag and Zn in Ag–ZnO nanocomposite (2 /H9262g/mL) were
0.621 and 0.626 /H9262g/mL, respectively. The results clearly signify
the presence of almost equal concentrations of Ag and Zn in the
synthesized nanocomposite and support the FE-SEM EDX findings.
3.2. Antibacterial activity of Ag–ZnO nanocomposite
3.2.1. Estimation of MIC and MKC of the as-synthesized
nanocomposite
The antibacterial studies were performed using S. aureus and
GFP expressing antibiotic resistant E. coli strains. The MIC and MKC
values for the Ag–ZnO nanocomposite [Zn:Ag (1:0.88)] were esti-
mated by monitoring the growth of S. aureus and GFP E. coli. Visual
turbidity analysis showed that 550 /H9262g/mL concentration of the
Ag–ZnO nanocomposite strongly inhibited the growth of GFP E. coli
cells and this was considered as MIC value. Upon re-inoculation into
fresh LB broth medium with 550 /H9262g/mL and 600 /H9262g/mL of Ag–ZnO
nanocomposite, no growth was observed for the nanocomposite
with 600 /H9262g/mL, which was thus considered as MKC value. Sim-
ilar experiments were carried out for determination of MIC and
MKC of Ag–ZnO nanocomposite against S. aureus . Lu et al. reported
the tyrosine-assisted synthesis of Ag–ZnO nanocomposites and its
synergistic antibacterial effect against E. coli and S. aureus based on
disk diffusion assay. The MIC of their Ag–ZnO nanocomposite is 600
and 400 /H9262g/mL for E. coli and S. aureus , respectively [18]. However,
the MIC value of our Ag–ZnO nanocomposite was found out to be
550 /H9262g/mL and 60 /H9262g/mL for E. coli and S. aureus respectively which
is less than the previously reported. Also, in a separate experiment
mixture of Ag and ZnO nanopowders were used as a control tocompare the additive effect of these individual nanopowders with
Ag–ZnO nanocomposite against antibiotic resistant GFP expressing
E. coli. After 12 h of incubation the bacterial growth was evaluated
by visual turbidity analysis. No growth was observed in all the tubes
except for the control bacteria. However, upon re-inoculation into
fresh medium growth was observed in the tubes containing mix-
ture of Ag and ZnO nanopowders similar to the control bacteria.
On the contrary, tube inoculated with Ag–ZnO nanocomposite MIC
concentrations showed very little growth but the tube with MKC
lacked growth (Supplementary Fig. S1). This experiment clearly
suggests the strong bactericidal potential of Ag–ZnO nanocom-
posite as an entirety compared to mixture of individual Ag and
ZnO nanopowders. Hence, our nanocomposite can serve as a more
effective and reliable nanoweapon against pathogenic and antibi-
otic resistant bacteria for food packaging applications and also to
combat hospital-related infections.
3.2.2.
Optical density measurements
The growth behavior of S. aureus and GFP E. coli in the pres-
ence of different concentrations of Ag–ZnO nanocomposites was
studied to correlate the observed bacterial density at 600 nm with
the antibacterial efficacy of the nanocomposites. Fig. 3(a and b)
represents the effect of different concentrations of nanocompos-
ite on growth of S. aureus and GFP E. coli, respectively. A marginal
decrease in the growth of E. coli and S. aureus was observed up
to 500 /H9262g/mL and 40 /H9262g/mL of nanocomposite, respectively. How-
ever, the growth was completely suppressed at 550 /H9262g/mL and
600 /H9262g/mL of nanocomposite in case of GFP E. coli and at 60 /H9262g/mL
and 70 /H9262g/mL of nanocomposite in case of S. aureus as compared
to untreated bacteria. Also, number of moles of Ag and Zn in the
obtained MIC and MKC values of Ag–ZnO nanocomposite against
GFP E. coli and S. aureus were calculated and are depicted in Table
S2. As obtained from the MIC and MKC measurements and opti-
cal density analysis implicates that the formulated heterostructure

I. Matai et al. / Colloids and Surfaces B: Biointerfaces 115 (2014) 359 – 367 363
Fig. 3. Effect of different concentrations of Ag–ZnO nanocomposite [Zn:Ag (1:0.88)] on growth of (a) S. aureus (n = 3) and (b) recombinant GFP E. coli (n = 3).
with ZnO on its surface exhibit preferentially stronger bactericidal
activity toward S. aureus and Ag exerts strong antibacterial activity
toward antibiotic resistant GFP E. coli.
3.2.3.
Fluorescence spectroscopic and microscopic analysis
Expression of GFP protein in E. coli cells facilitates rapid moni-
toring of antibacterial phenomena of Ag–ZnO nanocomposites by
spectroscopic and microscopic techniques. The fluorescence spec-
tral studies revealed continual decrease in fluorescence of GFP E.
coli with increase in the concentration of Ag–ZnO nanocomposites.
Strong green emission band at 510 nm was observed in untreated
bacteria which is the characteristic feature of healthy GFP E. coli.No fluorescence was observed in GFP E. coli treated with 550 /H9262g/mL
and 600 /H9262g/mL of nanocomposite suggesting complete eradication
of bacteria at these concentrations (Supplementary Fig. S2).
The fluorescence microscopic images (100× magnification)
of GFP E. coli treated with different concentrations of Ag–ZnO
nanocomposites are depicted in Fig. 4a. Significant decrease in
the GFP E. coli bacterial count was visualized with increase in
the concentration of nanocomposite. When the concentration of
nanocomposite was increased from 200 to 600 /H9262g/mL, a steady
decrease in the size and population of bacteria was observed
as compared to the control (untreated bacteria). Appearances of
flower shaped structures in the background of microscopic images
Fig. 4. (a) Fluorescence micrograph images of GFP E. coli treated with different concentrations of Ag–ZnO nanocomposite [Zn:Ag (1:0.88)], (i) untreated GFP E. coli, (ii)
200 /H9262g/mL Ag/ZnO, (iii) 300 /H9262g/mL Ag/ZnO, (iv) 400 /H9262g/mL Ag/ZnO, (v) 500 /H9262g/mL Ag–ZnO, (vi) 550 /H9262g/mL Ag/ZnO, (vii) 600 /H9262g/mL Ag/ZnO, (viii) only Ag–ZnO in LB medium.
All
images were taken at 100× magnification. (b) Gram stained micrograph images of S. aureus treated with different concentrations of Ag–ZnO nanocomposite [Zn:Ag
(1:0.88)],
(i) S. aureus control, (ii) 20 /H9262g/mL Ag/ZnO, (iii) 30 /H9262g/mL Ag/ZnO, (iv) 40 /H9262g/mL Ag/ZnO, (v) 50 /H9262g/mL Ag–ZnO, (vi) 60 /H9262g/mL Ag/ZnO, (vii) 70 /H9262g/mL Ag/ZnO, (viii)
only
Ag–ZnO in NB medium. All images were taken at 20× magnification.

364 I. Matai et al. / Colloids and Surfaces B: Biointerfaces 115 (2014) 359 – 367
Fig. 5. AFM images of (a) untreated (b) Ag–ZnO nanocomposite treated GFP E. coli (d) untreated and (e) treated S. aureus (scan field area 5 /H9262m × 5 /H9262m). FE-SEM micrographs
of treated (c) GFP E. coli and (f) S. aureus .
correspond to sugar moieties present in the bacterial growth
medium. The obtained fluorescence microscopic images corrob-
orate well with the fluorescence spectroscopic measurements
suggesting strong bactericidal potential of the as-synthesized
nanocomposites against GFP expressing antibiotic resistant E. coli.
Since S. aureus lacks any intrinsic fluorescence, traditional Gram’s
staining procedure was adopted for its visualization (Fig. 4b). The
presence of large number of grape-like structures confirms the
presence of S. aureus . From the stained micrographs it was noticed
that with the increase in concentration of nanocomposites from 20
to 70 /H9262g/mL, the number of bacteria went down with minimal in
60 /H9262g/mL and complete eradication in 70 /H9262g/mL. Depletion in the
bacterial count upon addition of nanocomposites suggests either
the inability of bacteria to grow in the presence of nanocomposites
or probably bacterial cell death.
3.3. Antibacterial mechanism of Ag–ZnO nanocomposite
3.3.1. AFM, FE-SEM and TEM analysis
To understand the antibacterial mechanism, the interaction of
Ag–ZnO nanocomposite with S. aureus and GFP E. coli bacteria andthe subsequent changes of cell morphology were studied by using
AFM, FE-SEM and TEM. Fig. 5 shows the representative AFM images
for the films of (a) untreated GFP E. coli and (b) Ag–ZnO nanocom-
posite treated GFP E. coli. (d) Untreated S. aureus (e) Ag–ZnO
nanocomposite treated S. aureus .
Initially GFP E. coli bacteria appeared as intact rods with no evi-
dence of membrane rupture and collapse (Fig. 5a). However, upon
treatment with Ag–ZnO nanocomposites, the bacteria demon-
strated strong evidence of membrane disorganization with greater
roughness as compared to the smooth surface of untreated E. coli
bacteria (Fig. 5b). Similarly, treated S. aureus (Fig. 5e) showed pro-
found membrane damage and distortion with increased roughness
contrary to the smooth contour of the control S. aureus (Fig. 5d). The
obtained FE-SEM micrographs of treated GFP E. coli and S. aureus
were in accordance with the AFM results. Treated GFP E. coli (Fig. 5c)
showed extensive membrane damage and leak-out of the intracel-
lular components causing shrinkage of cell and finally cell lysis.
Similarly, extensive membrane lysis and cell death of treated S.
aureus was observed (Fig. 5f). The damaged morphology and cell
rupture can be attributed to the binding and internalization of
Ag–ZnO nanocomposites. Taken together, these AFM and FE-SEM

I. Matai et al. / Colloids and Surfaces B: Biointerfaces 115 (2014) 359 – 367 365
Fig. 6. TEM micrographs of treated E. coli represent (a) attachment and internalization of Ag–ZnO nanocomposite and (b) cell lysis. Similar results were obtained for Ag–ZnO
nanocomposite treated S. aureus (c and d).
images are clear evidence for the antibacterial effects of Ag–ZnO
nanocomposites.
Fig. 6 represents the TEM images of treated E. coli and S.
aureus . Attachment and internalization of Ag–ZnO nanocompos-
ites onto bacterial cell walls was evident from the obtained TEM
micrographs. Considerable damage upon extensive binding to the
bacterial cell membrane (Fig. 6a) and significant reduction in the
size of E. coli rods due to shrinkage and lysis of the membrane
was observed (Fig. 6b). In addition, interactions of the nanocom-
posites with the cell wall resulted in enhanced permeability of the
membrane, allowing their entry into the cell and thereby possibly
causing cell death.
Fig. 6c represents the binding of the nanocomposite to the
grape-like S. aureus cell and Fig. 6d depicts the complete cell rupture
and death upon physical attack of the nanocomposite.
3.3.2.
Effect of Ag–ZnO nanocomposite on plasmid DNA
In order to study the effect of Ag–ZnO nanocomposite on
plasmid DNA (pDNA), the pDNA isolated from the untreated and
treated E. coli cells were analyzed by agarose gel electrophoresis
(Supplementary Fig. S3). The intensity of pDNA band in lane 2
corresponding to E. coli treated with 550 /H9262g/mL (MIC concen-
tration) of Ag–ZnO nanocomposite was significantly lower as
compared to control (untreated) pDNA in lane 1, suggests that
Ag–ZnO nanocomposite exert considerable effects on the plasmid
DNA replication leading to bacterial cell death. E. coli treated
with 400 /H9262g/mL of Ag–ZnO nanocomposite (i.e. less than MICconcentration) suggests that the bacteria are able to grow but
the pDNA (in lane 3) concentration is less than the control due
to induced cell death. TEM (Fig. 6) and electrophoretic data (Sup-
plementary Fig. S3) suggest the dual-mode antibacterial action
of the Ag–ZnO nanocomposites by damaging cell membrane and
inhibiting pDNA replication results in bacterial cell death.
3.3.3.
Ion release studies
The release of Ag+and Zn2+ions from Ag–ZnO nanocompos-
ite in water was analyzed by AAS after 6, 12 and 24 h incubation
times. The experiments were done in triplicate and mean was
taken for the obtained measurements. The results obtained are
depicted in Table S3. An increase in the Zn ion release was seen
with increment in the incubation times from 6 to 24 h for both
MIC and MKC concentrations. Similarly, the rate of Ag ion release
was found to increase from 6 h to 12 h; however a slight differ-
ence was seen in the release from 12 to 24 h in both the cases.
There was an obvious variation in release of these ions for MIC
and MKC values owing to the difference in the initial concentra-
tions. The presence of Ag and Zn ions in the water upon contact
with the nanocomposite strongly suggests the role of these in
inducing bacterial cell death either due to strong electrostatic inter-
action with the negatively charged cell membrane of the bacterial
cells or via production of intracellular reactive oxygen species.
These findings are in agreement with previously reported literature
[19–21] .

366 I. Matai et al. / Colloids and Surfaces B: Biointerfaces 115 (2014) 359 – 367
Fig. 7. Schematic representation of antibacterial mechanism of Ag–ZnO nanocomposite.
3.3.4. Antibacterial mechanism
Although the antibacterial effects of Ag and Zn NPs against var-
ious bacterial systems are well established, however very little is
known about their bactericidal mechanism at molecular level and
thus mode of action. Here is an attempt to explore the antibacterial
mechanism of Ag–ZnO nanocomposites on the basis of our inves-
tigation and by taking into consideration of the previous proposed
findings (Fig. 7).
There could be four plausible ways of antibacterial activity of
Ag–ZnO nanocomposite responsive for bacterial cell death.
(i) Ag–ZnO nanocomposite may interact directly with the bac-
terial cell membrane [19] by release of silver (Ag+) and zinc
ion (Zn2+) upon surface oxidation [20] or via porosity of com-
posite (as supported by our AAS and TEM analysis) or by
electrostatic interactions between the ions released and the
negatively charged bacterial cell wall [21].
(ii) Disruption of the bacterial cell membrane (as supported by our
TEM, FE-SEM and AFM analysis) would be the other probable
mode of nanocomposite action, as it paves the way into the
bacterial cells by leading to membrane protein and lipid bilayer
damage as reported in previous studies [22]. The damage could
be either by release of Ag+/Zn2+ions or by formation of ROS
[5,23] .
(iii) At cellular level the disruption of bacterial cell membrane, by
either altering the membrane protein or enzyme activity in
a ROS mediated manner [22]. At molecular level it inhibits
DNA/plasmid replication and proteins/enzymes in cells [24]
either via ROS formation or by Ag+/Zn2+ions directly [4]. Inhi-
bition of DNA replication by Ag+/Zn2+ions [4,24] by interacting
with sugar phosphate groups or gene alteration, thus affecting
necessary protein expression for cellular functioning [25]; Ag+
ion is known for its interaction with ribosomal subunit 30S,
thus leading to inhibition of the protein synthesis necessary
for bacterial machinery [26].
(iv) Leakage of intracellular material due to membrane disruption
may cause shrinkage of the cell membrane, ultimately leading
to cellular lysis as justified by our TEM micrographs (Fig. 6).4. Conclusion
In summary, the present study, for the first time, reports the
rapid synthesis of Ag–ZnO nanocomposites with nearly equal wt%
of Ag and Zn based on simple microwave reactions in absence of
any surfactants. Since, no surfactant has been utilized in the syn-
thesis procedure the chances of artifacts are significantly reduced.
The toxicity induced by silver and zinc compounds is long known
yet their precise interactions with the bacterial cells are less
understood. For instance, AgNO 3exhibits antimicrobial activity
with MIC of 3 /H9262M (323 /H9262g/L−1) against E. coli [27]. However, the
cytotoxic effects of AgNO 3and increasing bacterial resistance to
current antimicrobial compounds have emphasized the use of sil-
ver nanoparticles (Ag NPs) that demonstrate controlled killing
with comparably reduced cytotoxicity. Proper understanding of
the nanoparticle toxicity with the prokaryotic systems is extremely
significant before their discharge in water and soil bodies and for
various biomedical applications. For instance, Sinha and cowork-
ers studied the interaction and nanotoxic effect of ZnO and Ag
nanoparticles at millimolar levels (2–10 mM) on mesophilic and
halophilic bacteria spp. such as Enterobacter , Bacillus subtilis and
Marinobacter [19]. Rathnayake et al. and Hoseinzadeh et al. reported
strong antibacterial properties of nanosized ZnO against E. coli and
S. aureus [28,29] . ZnO NPs at concentrations 1250 and 5000 /H9262g/mL
were found to kill E. coli and S. aureus respectively [29]. Bacteri-
cidal activity of Ag NPs has been detected at low concentrations
against many bacterial strains. Guzman et al. reported bacteri-
cidal activity of Ag NPs against E. coli, S. aureus and Pseudomonas
aeruginosa at ∼216 ppm (parts per million) [30]. Matzke et al. found
that Ag NPs inhibited Pseudomonas putida growth at 3.4 /H9262g/L con-
centration [31]. Genipin-crosslinked chitosan/polyethylene glycol
(GC/PEG)ZnO(0.02 g)/Ag(1 mg) nanocomposites have shown to
inhibit the growth of E. coli, P. aeruginosa , B. subtilis and S. aureus
upon addition and increasing content of ZnO and Ag nanoparticles
in the matrix [32]. Recently, Motshekgya and coworkers adopted
microwave assisted approach to prepare Ag–ZnO NPs supported on
bentonite clay and tested its antibacterial activity against Entero-
coccus faecalis and E. coli. ZnO (0.5 g)-clay itself showed inhibition

I. Matai et al. / Colloids and Surfaces B: Biointerfaces 115 (2014) 359 – 367 367
of bacterial growth which further enhanced upon introduction of
Ag NPs [33]. Most of the antibacterial studies done so far are based
on disk diffusion which serves as a qualitative approach and may
sometime give false positive results. Extent of antibacterial activity
by a nanoparticle depends on several parameters, such as method
of preparation, capping agents and its size. In the present study we
have formulated a nanostructure using microwave reactions that
combines the antibacterial properties of Ag and ZnO NPs in a single
platform competent to induce sustained and pronounced antibac-
terial activity against S. aureus and antibiotic resistant E. coli with
60 and 550 /H9262g/mL as MIC concentrations, respectively, which is less
than the previously reported. We have quantitatively assessed the
antibacterial properties of these Ag–ZnO nanocomposites against
antibiotic resistant bacteria and suggest their applicability toward
development of antimicrobial packages and for biomedical appli-
cations like wound dressings.
Certain polymeric matrices can be modified by introducing
Ag–ZnO nanocomposites as a new approach for preservation and
enhancement of shelf life of the constituents by reducing the
adhesion of detrimental microbes. One such effort has been made
by modification of low-density polyethylene (LPDE) films consti-
tuting Ag and ZnO nanoparticles for fresh orange juice packing
[34,35] . Various catheter-associated infections can be prevented
by adding Ag–ZnO nanocomposites to traditional polymers, such as
polyurethane and silicone [36]. In addition, coating surgical masks
and instruments with such nanocomposite can significantly retard
microbial adhesion to prevent nosocomial infections [37]. Thus, the
present study established a novel and simple method to synthe-
size Ag–ZnO nanocomposites and also investigated its antibacterial
mechanism. Such understanding of antibacterial mechanism would
primarily establish Ag–ZnO nanocomposites as a potent broad-
spectrum antibacterial agent for its wide applications in various
fields.
Acknowledgements
Our sincere thanks to Department of Science and Technology
(Water Technology Initiative), Uttarakhand State Biotechnology
Department, and Ministry of Human Resource Development
(Faculty Initiation Grant, IIT Roorkee), Government of India,
for the financial support. Institute Instrumentation Centre and
Department of Chemistry, Indian Institute of Technology Roor-
kee are sincerely acknowledged for providing various analytical
facilities.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.colsurfb.
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