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Contents lists available at ScienceDirect

Journal of Industrial and Engineering Chemistry

journal homepage: www.elsevier.com/locate/jiec
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Biologically synthesized silver nanoparticles enhances antibiotic

activity against Gram-negative bacteria
Q1 Sangiliyandi
a
b

Gurunathan a,b,*

Dept of Animal Biotechnology, Konkuk University, 1 Hwayang-Dong, Gwangin-gu, Seoul 143-701, South Korea
GS Institute of Bio and Nanotechnology, Coimbatore, Tamilnadu, India

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 25 September 2014
Received in revised form 5 March 2015
Accepted 2 April 2015
Available online xxx

Here we report a simple, fast, cost-effective, and nonpolluting approach for synthesis of silver
nanoparticles (AgNPs) using leaf extract of Typha angustifolia. We demonstrate the dose-dependent
antibacterial activity of AgNPs and different antibiotics against Escherichia coli and Klebsiella pneumoniae.
Furthermore, we demonstrate the efcacy of AgNPs in combination with various broad-spectrum
antibiotics against E. coli and K. pneumoniae. The results show that combinations of antibiotics and AgNPs
show signicant antimicrobial effects at sub-lethal concentrations of the antibiotics. These data suggest
that combinations of antibiotics and AgNPs can be used therapeutically for the treatment of infectious
diseases.
2015 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering
Chemistry.

Keywords:
Antibacterial activity
Antibiotics
Escherichia coli
Klebsiella pneumoniae
Typha angustifolia
Q3 Silver nanoparticles

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Introduction

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Recently, nanotechnology has emerged as one of the fastest

growing areas of science and technology. Nanoparticles have
generated much interest in academia as well as industry because
they bridge the gap between bulk materials and atomic or
molecular structures [1]. Owing to their unique properties,
nanomaterials are increasingly being used in commercial applications in a variety of elds, including optics, electronics, magnetics,
mechanics, catalysis, energy science, nanobiotechnology, and
nanomedicine, particularly as antimicrobial agents for diagnostic
purposes [1]. In addition, silver nanoparticles (AgNPs) are
extensively used for the production of clothing, catheters, electric
home appliances, and biomedical implants [2]. Because of their
well-known antiseptic activities, silver compounds are used in
clinical settings to prevent skin infections, such as in the treatment
of burns (e.g., silver sulfadiazine) and as coatings on various
surfaces such as catheters [2,3]. Furthermore, nanoparticles
possess dimensions below the critical wavelength of light. This
renders them transparent, a property that makes them very useful

* Corresponding author at: Department of Animal Biotechnology, Konkuk

University, 1 Hwayang-dong, Gwangin-gu, Seoul 143-701, South Korea.
Tel.: +82 2 450 0457; fax: +82 2 458 5414.
E-mail addresses: gsangiliyandi@yahoo.com, lvsangs@yahoo.com

for applications in cosmetics, coatings, packaging, and diagnostics

[4]. Because of the high demand, one trillion dollars worth of
nanotechnology-based products is expected on the market by the
year 2015 [5].
The most widely used methods for synthesis of metallic
nanoparticles are traditional physical and chemical methods.
Conventional physical methods tend to yield low amounts of
nanoparticles, while chemical methods are often toxic, consume a
lot of energy, and require the use of stabilizing agents such as
sodium dodecyl benzyl sulfate or polyvinyl pyrrolidone (PVP) to
prevent agglomeration of the nanoparticles [4,6]. Therefore, a costeffective, simple, rapid, high-yield, and environmentally friendly
approach for the synthesis of metallic nanoparticles is needed.
Among several possible approaches, biological synthesis of
nanoparticles is particularly promising owing to the ready
availability of resources including viruses, bacteria, fungi, algae,
plants, and plant products [4].
Recently, several microorganisms have been exploited for
synthesis of silver and gold nanoparticles. For example, a silverresistant bacterial strain isolated from silver mines, Pseudomonas
stutzeri AG259, accumulates AgNPs within the periplasmic space
[7,8]. Parikh et al. [9] found that Morganella sp. RP-42 produced
extracellular crystalline AgNPs of 20  5 nm when exposed to silver
nitrate. Lactobacillus spp. produced microscopic gold, silver, and gold
silver alloy crystals of well-dened morphology when exposed to
high concentrations of metal ions [10]. Bacillus licheniformis produces

http://dx.doi.org/10.1016/j.jiec.2015.04.005
1226-086X/ 2015 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry.

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AgNPs with an average size of 50 nm both intracellularly [11] and

extracellularly [12]. Sweeney et al. [13] demonstrated that Escherichia
coli spontaneously formed cadmium sulde semiconductor nanocrystals when incubated with cadmium chloride and sodium sulde.
E. coli also produces AgNPs with an average size of 50 nm [14].
Kowshik et al. [15] demonstrated that MKY3, a silver-tolerant yeast
species, produced AgNPs ranging in size from 2 to 5 nm, and
Mukherjee et al. [16] described the synthesis of intracellular AgNPs
using the fungus Verticillium sp. In addition, plants have been used in
the synthesis of nanoparticles. Shankar et al. [17] reported the
extracellular synthesis of AgNPs by reduction of aqueous Ag+ ions
using extract of geranium leaves, and extract from lemongrass
(Cymbopogon exuosus) was used to synthesize triangular gold
nanoprisms [18]. Interestingly, the synthesis of AgNPs using plant
extracts is fairly rapid compared with synthesis using bacteria or
fungi [17]. However, few plants have been exploited for the synthesis
of silver or gold nanoparticles. Therefore, we attempted to use a
previously unexplored species, Typha angustifolia, for AgNP synthesis.
T. angustifolia is a monocot found in tropical and temperate regions of
the world in marshes and wetlands of various depths. It is a common
plant of wetlands and is an unexploited taxon that can be used as a
source of food, medicines, and bers as well as for the synthesis of
nanomaterials. Londonkar et al. [19] reported that crude aqueous
extracts of the aerial part of T. angustifolia plants contained alkaloids,
tannins, steroids, phenols, saponins, and avonoids. Based on the
presence of these compounds, we expected that the proteins,
polysaccharides, or secondary metabolites of T. angustifolia leaf
extracts would reduce Ag+ ions to the Ag0 state, resulting in the
formation of silver nanoparticles. Recently, Singhal et al. [20]
synthesized AgNPs using Ocimum sanctum leaf extract and found
that these nanoparticles showed signicant antibacterial activity
against E. coli and Staphylococcus aureus. Although several studies
have demonstrated the antibacterial activity of AgNPs, studies of the
combination of AgNPs and antibiotics are warranted.
Several studies have shown not only an increasing number of
infections caused by gram-negative bacteria worldwide but also
mounting rates of resistance. In a study of 1265 intensive care units
in 75 countries, Vincent et al. [21] found that gram-negative
bacteria were present in 62% of patients with an infection, while
gram-positive bacteria were present in 47% of these patients.
Gram-negative bacteria are highly adaptive pathogens that can
develop resistance to antibiotics through several mechanisms; this
resistance is a serious concern in terms of public health and health
care costs [2226]. Gram-negative bacteria are common causes of
intra-abdominal infections, urinary tract infections, nosocomial
pneumonia, and bacteremia [27]. Tamma et al. [28] reported that
combination antibiotic therapy may have benets other than the
prevention of resistance during denitive treatment. We selected
E. coli and Klebsiella pneumoniae as model gram-negative bacteria
because both bacteria cause infections in the abdominal and
urinary tracts. In light of the observations described above, we rst
investigated the extracellular synthesis of AgNPs using leaf extract
of T. angustifolia. Second, we investigated the antibacterial effect of
the prepared AgNPs against E. coli and K. pneumoniae. Finally, we
investigated the effect of combinations of selected antibiotics with
AgNPs against E. coli and K. pneumoniae.

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Materials and methods

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Reagents, bacterial strains, and culture conditions

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Mueller Hinton Broth (MHB), Mueller Hinton Agar (MHA), silver

nitrate, gentamicin, cefotaxime, and meropenem were purchased
from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were
purchased from Sigma-Aldrich unless otherwise stated. The E. coli

and K. pneumoniae strains used in the present study were from our
culture collections.
Bacterial culture and media preparation were carried out
according to previously described methods [14]. Briey, E. coli and
K. pneumoniae were grown aerobically at 37 8C in MHB. The
cultures were maintained by streaking the organisms on LB agar
plates and subculturing every fortnight. Pure colonies were
isolated and stored at 80 8C. Cells were harvested by centrifugation at 6000 rpm for 10 min and resuspended in sterile LB medium
to obtain an optical density at 600 nm of 1.0.

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Synthesis of AgNPs

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T. angustifolia leaves were collected from a marshy area around

Coimbatore, Tamilnadu, India, and stored at 4 8C until needed.
Twenty grams of leaves were washed thoroughly with doubledistilled water and then sliced into ne pieces, approximately
15 cm2, using a sharp stainless steel knife. The nely cut leaves
were suspended in 100 mL of sterile distilled water and boiled for
5 min. The resulting mixture was ltered through Whatman lter
paper (grade No. 1). The ltered extract was used for the synthesis
of AgNPs by adding 10 mL of extract to 100 mL of 1 mM aqueous
AgNO3 solution and stirring the mixture at 37 8C for 15 min. The
bioreduction of AgNO3 was monitored spectrophotometrically at
420 nm.

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Characterization of AgNPs

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The synthesized nanoparticles were characterized according to

previously described methods [14]. Briey, the prepared AgNPs
were characterized primarily by UVvis spectroscopy, which has
proved to be a very useful technique for the analysis of AgNPs.
UVvis spectra were obtained using a Biochrom (Cambridge, UK)
WPA Biowave II UVvis spectrophotometer. The synthesized AgNPs
were freeze-dried, powdered, and analyzed by X-ray diffraction
(XRD) spectroscopy. The spectra were produced using an XPertMPD X-ray diffractometer (Philips, the Netherlands) and Cu Ka
radiation (l = 1.5405 A) over an angular range of 10 to 808 at 40 kV
and 30 mA. The dried powder was diluted with KBr at a ratio of 1:100
and analyzed by Fourier transform infrared spectroscopy (FTIR)
using a Spectrum GX spectrometer (Perkin Elmer Inc., USA) within
the range of 500 to 4000 cm 1. The size distribution of the dispersed
particles was measured using a Zetasizer Nano ZS90 (Malvern
Instruments Ltd., UK). Transmission electron microscopy (TEM) was
used to determine the size and morphology of the AgNPs. A small
amount of aqueous dispersion was dropped on copper grids, which
were dried and examined in the transmission electron microscope
(JEM-1200EX).

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Determination of minimum inhibitory concentrations of AgNPs and

antibiotics

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To determine the minimum inhibitory concentrations (MICs) of

AgNPs and antibiotics, bacterial strains were cultured in MHB. Cell
suspensions were adjusted to obtain standardized populations
by measuring the turbidity with a spectrophotometer (DU530,
Beckman, Fullerton, CA, USA). Susceptibility tests were performed
by twofold microdilution of the antibiotics and AgNPs in standard
broth according to the Clinical and Laboratory Standards Institute
(CLSI) guidelines (CLSI, 2003). The bacterial strains were grown in
MHB to mid-log phase (1  106 cells/mL) and diluted in fresh MHB,
and 0.1 mL of the diluted cell suspension was dispensed into each
well of a 96-well microtiter plate. E. coli and K. pneumoniae were
then exposed to different concentrations of AgNPs or antibiotics.
Growth was assayed by monitoring absorbance at 600 nm using a
microplate reader (EMax, Molecular Devices, Sunnyvale, CA, USA).

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The MICs of the AgNPs and antibiotics were determined as the

lowest concentrations that inhibited visible growth of the bacteria.
Antibiotic or AgNP concentrations that reduced the number of
susceptible cells by less than 20% after 24 h of incubation were
designated as sublethal. Viability assays were carried out with
different concentrations of antibiotics or AgNPs alone or with
combinations of sublethal concentrations of antibiotics and AgNPs.

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Disc diffusion assay

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An agar diffusion assay was performed as described previously,

using MHA [29,30]. Conventional and broad-spectrum antibiotics
were selected to assess the effect of combined treatment with
antibiotics and AgNPs. Based on the CLSI standard, the following
concentrations of antibiotics were used: gentamicin, 10 mg/mL;
cefotaxime, 30 mg/mL; and meropenem, 10 mg/mL. For the combination treatments, each standard antibiotic disc was impregnated
with 1.4 mg of AgNPs. A single colony of each test strain was grown
overnight in MHB on a rotary shaker (200 rpm) at 37 8C. The inocula
were prepared by diluting the overnight cultures with 0.9% NaCl to a
0.5 McFarland standard. Inocula were applied to the plates along
with control discs and discs containing different antibiotics. Similar
experiments were carried out with AgNPs alone. After incubation at
37 8C for 24 h, zones of inhibition (ZOIs) were determined by
subtracting the disc diameter from the diameter of the total
inhibition zone. The assays were performed in triplicate. Antibacterial activity was quantied by the equation (AB)/A  100, where A
and B are the ZOIs for antibiotic and antibiotic with AgNPs,
respectively.

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In vitro killing assay

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An in vitro killing assay was performed as described previously

[31] with suitable modications. Cells were grown overnight in
MHB at 37 8C and then regrown in fresh medium for 4 h before
being collected by centrifugation and suspended in saline. A cell
suspension consisting of 106 cells/mL was incubated with various
concentrations of antibiotics, AgNPs, or combinations of AgNPs
with an antibiotic and incubated at 37 8C without shaking. Aliquots
(100 mL) were withdrawn at specic time intervals and spread on
MHA plates undiluted or after 10-fold serial dilution to determine
the number of colony forming units (CFUs). Experiments were
performed with various controls including a positive control
(AgNPs and MHB, without inoculum) and a negative control (MHB
and inoculum, without AgNPs). All samples were plated in
triplicate and values were averaged from three independent
experiments. The experiments with sublethal concentrations of
antibiotics or AgNPs, or combinations of AgNPs and antibiotics,
were performed for 4 h at 37 8C.

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Measurement of reactive oxygen species generation

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Reactive oxygen species (ROS) generation was measured as

described earlier [32]. A quantitative assay for superoxide anions
was carried out using an in vitro toxicology assay kit based on XTT
sodium salt (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt; catalog number Tox-2) purchased from Sigma-Aldrich, according to the manufacturers
instructions. E. coli and K. pneumoniae were grown in MHB in the
presence of meropenem, AgNPs, or a combination of meropenem
and AgNPs. Cells were then washed with phosphate-buffered saline
(PBS) and resuspended in PBS at a concentration of 2106 viable cells
(determined as CFUs) per milliliter. XTT was added to the cell
suspension at a concentration of 125 mM from a stock solution
(7.5 mM) prepared in PBS. Cell suspensions were incubated at 30 8C
on a rotary shaker for 24 h, aliquots were spun in a microfuge, and

the absorbance of the supernatant was measured at 450 nm. XTT

reduction in the absence of cells was always determined as a control
and subtracted from the values observed in the presence of cells.

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Statistical analysis

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All experiments were carried out in triplicate and repeated at

least three times. The results are presented as means  SD. All
experimental data were compared using Students t test. A p-value
less than 0.05 was considered statistically signicant.

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Results and discussion

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Synthesis and characterization of AgNPs

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To synthesize AgNPs, the leaf extract from T. angustifolia was 245

used as both reducing and stabilizing agent. In a typical reaction, 246
10 mL of T. angustifolia leaf extract was added to nal volume 247
100 mL contains of 1 mM aqueous AgNO3 solution with 10 mL of 248
leaf extract, and the mixture was stirred with a magnetic stirrer at 249
room temperature for 15 min. The green mixture of silver nitrate 250
and leaf extract changed rapidly after 15 min at 37 8C to a brown 251
suspension, whereas silver nitrate without leaf extract showed no 252
color change (Fig. 1 inset). This indicated that AgNPs can be 253
synthesized using T. angustifolia leaf extract. Aqueous extracts of 254
leaves of T. angustifolia are known to contain alkaloids, tannins, 255
steroids, phenols, saponins, and avonoids, and these secondary 256
metabolites could induce the formation of nanoparticles by 257
serving as reducing agents. Chandran et al. [33] synthesized 258
silver nanoparticles by incubating Aloe vera leaf extract with 259
silver nitrate for 24 h. Interestingly, in our experiment the color 260
change was observed within 15 min of incubation; however, the 261
reaction was carried out for 30 min. The reduction of silver ions 262
using leaf extract occurs more rapidly than with bacterial culture 263
[11,14].
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We conrmed the synthesis of AgNPs using UVvis spectros- 265
copy. The strong surface plasmon resonance (SPR) of the AgNPs 266
produced a peak centered near 420 nm. The SPR band at 420 nm 267
indicated that formation of AgNPs had occurred (Fig. 1). Mulvaney 268
(1996) reported that AgNPs exhibit a yellowish-brown color in Q4269
water owing to excitation of surface plasmon vibrations.
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Fig. 1. Synthesis and characterization of AgNPs produced using leaf extract of Typha
angustifolia. The inset image shows containers with samples of AgNO3 (1), leaf
extract (2), and a mixture of AgNO3 and leaf extract at 15 min (3) and 30 min (4)
after mixing. The color of the mixture changed from green to dark brown 15 min
after mixing, indicating the formation of AgNPs. The absorption spectrum of the
AgNPs exhibited a strong, broad peak at 420 nm. This band was attributed to the
surface plasmon resonance of the AgNPs. (For interpretation of the references to
color in this gure legend, the reader is referred to the web version of this article.)

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XRD analysis

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To conrm the crystalline nature of the AgNPs, XRD analysis

was performed. The XRD spectrum of the AgNPs showed four
intense peaks with 2u values of 23.618, 29.908, 33.878, and 46.738
(Fig. 2). The ve diffraction peaks in the standard pattern of silver
are indexed as reections of the (1 1 1), (2 0 0), (2 2 0), (3 1 1), and
(2 2 2) planes of face-centered cubic (fcc) silver (JCPDS le No. 040783). The (2 0 0), (2 2 0), (3 1 1), and (2 2 2) Bragg reections are
weak and broadened relative to the intense (1 1 1) reection. This
feature indicates that the nanocrystals are (1 1 1) oriented [17,34].
The Full Width at Half Maximum (FWHM) values measured for
1 1 1, 2 0 0, 2 2 0, and 3 1 1 planes of reection were used with the
DebyeScherrer equation to calculate the size of the nanoparticles.
The particle sizes obtained from XRD line broadening agreed well
with that obtained from TEM. From these the average particle size
was found to be around 8 nm. A comparison of our XRD spectrum
with the standard spectrum conrmed that the silver particles
formed in our experiment were in the form of nanocrystals, as the
peaks at 2u values of 23.618, 29.908, 33.878, and 46.738 corresponded
to reections of the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of fcc
silver, respectively. A few additional intense and unassigned peaks
were also observed in the area of the characteristic peaks of silver.
These additional and unidentied sharp Bragg peaks may have
resulted from bioorganic compounds or proteins present in the leaf
extract or may be related to amorphous and crystalline organic
phases on the surface of the nanoparticles. Similar results were
obtained for silver nanoparticles synthesized using geranium leaf
extract [17], Krishna tulsi (O. sanctum) leaf extract [35], Ipomoea
indica owers [36], carob leaf extract [37], and mushroom extract
[38].

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FTIR analysis of AgNPs

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FTIR measurements were used to identify the biomolecules in

the leaf extract that were responsible for the reduction of the Ag+
ions and the capping of the bioreduced silver nanoparticles [17].
After complete reduction of the Ag+ ions and formation of the
AgNPs, the silver nitrate solution was centrifuged at 10,000 rpm for
15 min to isolate the AgNPs from free proteins or other compounds
present in the solution, and the centrifugate was collected for FTIR
analysis [17]. Fig. 3A shows the FTIR spectra of the T. angustifolia
leaf extract. The FTIR spectra reveal the presence of different
functional groups. The IR bands observed at 3314 and 1635 cm 1 in

dried leaf are characteristic of the OH and C5

5O stretching modes
for the OH and C5
5O groups possibly of secondary metabolites
present in the leaf extract.
The FTIR spectrum of the AgNPs exhibited peaks at 1728 and
1635 cm 1, which could be assigned to ester C5
5O groups of
chlorophyll [17,40] and the amide I bond of proteins [20,41],
respectively. The water-soluble fraction of T. angustifolia leaves
contains large amounts of alkaloids, tannins, steroids, phenols,
saponins, and avonoids [19], and these secondary metabolites
could induce the formation of nanoparticles by serving as reducing
agents. It is possible that terpenoids also contribute to the
reduction of silver ions and, in the process, are oxidized to carbonyl
groups, resulting in the band at 1728 cm 1. Upon formation of the
AgNPs, the peak corresponding to the amide I band at 1635 cm 1
broadened, indicating capping of the AgNPs by proteins (Fig. 3B).
The absorption peak at 1635 cm 1 arises from the carbonyl stretch
in proteins, while the peak at 3409 cm 1 is due to OH stretching in
alcohols and phenolic compounds [20,41]. The absorption peak at
1635 cm 1 is close to that reported for native a protein [42], which
suggests that proteins are interacting with the biosynthesized
nanoparticles and that their secondary structure is not affected
during reaction with the Ag+ ions. It is known that proteins can
bind to gold nanoparticles through either free amine groups or
cysteine residues [43]. A similar mechanism is possible for silver
nanoparticles, since the leaf extract from T. angustifolia capped the
silver nanoparticles, thereby stabilizing them. Similar FTIR
patterns were observed for silver nanoparticles synthesized using
geranium leaf extract [17] or O. sanctum leaf extract [20,35].

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XPS analysis of AgNPs

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X-ray photoelectron spectroscopy (XPS) was utilized to

investigate the chemical state of the leaf extract of T. angustifolia
leaves mediated synthesis of AgNPs. The quantitative Ag/C atomic
ratios of the samples were determined using the peak area ratio of
the corresponding XPS core levels and the sensitivity factor (SF) of
each element in XPS [39]. Fig. 4 shows high resolution XPS spectra
of the C (1s) core level for the AgNPs. As shown in Fig. 4, two peaks
located at the binding energies of 368.4 and 373.6 eV were
observed, which conrming the successful formation of Ag(0) by
leaf extracts. The binding energies of Ag(3d5/2) and Ag(3d3/2)
peaks were found at binding energies of 368.4 and 373.6 eV,
respectively. To further understand the chemical state of the AgNPs
on the surface, a detailed deconvolution of the Ag(3d) peak was

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Fig. 2. XRD pattern of silver nanoparticles synthesized using T. angustifolia leaf extract.

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Fig. 3. FTIR pattern of T. angustifolia leaf extract (A) and silver nanoparticles synthesized using T. angustifolia leaf extract (B).

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also performed. The binding energy of the Ag(3d5/2) core level for
Ag, Ag2O, and AgO is 368.4, 368.3, and 367.5 eV, respectively. Based
on the Ag (3d5/2) peak analysis, we have found that about 94% of
the silver atoms on the surface were in the Ag0 (metallic) state,
while only about 1% and 5% of the silver atoms were in the Ag+ and
Ag2+ chemical states, respectively. These results are in good
agreement with earlier report suggested that synthesis of silver
using Allophylus cobbe leaf extracts [39].

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Dynamic light scattering analysis

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Dynamic light scattering (DLS) as a useful technique to evaluate

particle size, size distribution, and the zeta potential of nanomaterials in solution [14,38,39]. The characterization of nanoparticles
in solution before assessment of in vitro toxicity is a high priority

[44] because particle size, size distribution, particle morphology,

particle composition, surface area, surface chemistry, and particle
reactivity in solution can affect nanoparticle toxicity [44]. In the
present study, we used DLS in conjunction with TEM to evaluate
the size distribution of the synthesized silver nanoparticles. The
DLS pattern revealed that the AgNPs synthesized using T.
angustifolia leaf extract had an average size of 8  4 nm (Fig. 5).
Singhal et al. [20] reported that silver nanoparticles synthesized using
O. sanctum leaf extract had an average diameter of 22.38 nm.

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TEM analysis of AgNPs

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TEM is one of the most valuable tools for direct and accurate
analysis of the size and structure of nanoparticles. TEM has been
used previously to obtain essential information on primary

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Fig. 5. Size distribution analysis by DLS. The sizes of the AgNPs ranged from 2 to
15 nm. The average particle size was 8 nm.
Fig. 4. XPS spectra of silver nanoparticles synthesized using T. angustifolia leaf
extract.

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nanoparticle sizes and morphologies [44]. Therefore, we examined

the size and morphology of the AgNPs using TEM. The transmission
electron micrographs of the AgNPs revealed that the nanoparticles
were distinct, uniform, spherical, and well separated from each
other. The particle size was estimated from more than 200
particles in TEM images and was found to be between 3 and 18 nm,
with an average size of 8 nm (Fig. 6A). We also observed that the
AgNPs were evenly distributed in the analyzed sample. The particle
size distribution determined from TEM images is shown in
(Fig. 6B). Shankar et al. [17] reported that the size of nanoparticles
produced using geranium leaf extract ranged from 16 to 40 nm.
Interestingly, our data suggest that silver nanoparticles produced
using T. angustifolia leaf extract are smaller, which might result in
better antimicrobial activity.

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Determination of MICs and sub-lethal concentrations of AgNPs and

antibiotics

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The MICs of the AgNPs and antibiotics were dened as the

lowest concentrations that completely inhibited visible growth of
bacteria after incubation at 37 8C for 24 h. In this study, E. coli and
K. pneumoniae were used as model gram-negative bacteria to
evaluate the antibacterial activities of AgNPs and three different

antibiotics. Bacterial cells were incubated with different concentrations of AgNPs for 24 h in MHB. Medium without AgNPs was
used as a control. The E. coli and K. pneumoniae cell counts were
signicantly reduced by treatment with AgNPs in comparison with
the control. As shown in Table 1, the AgNPs showed similar MICs
(1.4 mg/mL) towards E. coli and K. pneumoniae. Size, surface area,
and surface functionalization are major factors that inuence the
biokinetics and toxicity of nanomaterials [45]. The antibiotics also
showed similar MICs for E. coli and K. pneumoniae: 1.0 mg/mL for
gentamicin, 0.5 mg/mL for cefotaxime, and 0.4 mg/mL for meropenem (Table 1).

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Dose-dependent antibacterial effect of AgNPs

412

We assessed the dose-dependent effect of AgNPs on E. coli and

K. pneumoniae, the relative susceptibility of both species to AgNPs,
and the extent of the bactericidal activity of AgNPs. Fig. 7 shows the
toxic effect of the biologically synthesized AgNPs on E. coli and K.
pneumoniae. The bacterial strains were treated with 8 nm AgNPs at
concentrations between 0.2 and 1.4 mg/mL. The introduction of
AgNPs reduced cell viability in comparison with the negative
control. Cell viability was reduced further as the concentration of
AgNPs increased. As determined earlier in the MIC experiment, no
visible bacterial growth was observed above an AgNP concentration of 1.4 mg/mL for both E. coli and K. pneumoniae (Fig. 7). These

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Fig. 6. Determination of AgNP size and shape using TEM. (A) The size and morphology of AgNPs were analyzed using TEM. The average particle size was 8 nm. (B) Particle size
distribution determined from TEM images.

Please cite this article in press as: S. Gurunathan, J. Ind. Eng. Chem. (2015), http://dx.doi.org/10.1016/j.jiec.2015.04.005

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Table 1
Determination of MICs and sub-lethal concentrations of antibiotics and AgNPs.
Bacterial species

MIC of various antibiotics and AgNPs (mg/ml)

GN

CE

MPM

AgNPs

E. coli
K. pneumoniae

1.0
1.0

0.5
0.5

0.40
0.40

1.4
1.4

Sub lethal concentration of antibiotics and AgNPs

(mg/ml)

E. coli
K. pneumoniae

GN

CE

MPM

AgNPs

0.25
0.25

0.15
0.15

0.10
0.10

0.3
0.3

Fig. 7. Effect of AgNPs on cell survival. E. coli and K. pneumoniae cells were incubated
with various concentrations of AgNPs. Bacterial survival was determined at 4 h by a
CFU count assay. The experiment was performed with various controls including a
positive control (AgNPs and MHB, without inoculum) and a negative control (MHB
and inoculum, without AgNPs). The results are expressed as the means  SD of three
separate experiments, each of which contained three replicates. Treated groups
showed statistically signicant differences from the control group by Students t test
(p < 0.05).

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results show that the AgNPs synthesized from T. angustifolia leaf

extract exhibited a strong, dose-dependent antimicrobial activity
against both test microorganisms (Fig. 7). The antimicrobial
activity of these AgNPs is greater than that of AgNPs from other
sources such as bacteria and fungi. Li et al. [46] reported that at a
concentration of 10 mg/mL, commercially prepared silver nanoparticles completely inhibited the growth of 107 CFUs/mL of E. coli

in liquid MHB. Anthony et al. [47] evaluated the toxicity of 40 nm

AgNPs prepared from culture supernatant of Bacillus marisavi and
found that the MIC against Pseudomonas aeruginosa was 10 mg/mL.
Several studies have examined the mechanisms underlying the
antimicrobial activity of AgNPs from various sources. Shrivastava
et al. [48] studied the interaction of silver nanoparticles with E. coli
and found that the nanoparticles adhered to the bacterial cell wall
and subsequently penetrated the cell, eventually killing the
bacteria by destroying the cell membrane. AgNPs may pass
through the cell wall of bacteria and oxidize surface proteins on the
plasma membrane, consequently disturbing cellular homeostasis
[45,49]. Several research groups have suggested that AgNPs may
attach to the surface of the cell membrane and disturb membrane
functions such as permeability and respiration [49,50]. Our results
suggest that AgNPs synthesized using a biological method, such as
the method used in this study, are smaller than nanoparticles
synthesized by other methods. Since smaller nanoparticles are
taken up by cells more easily than larger particles and have a larger
surface area available for interaction with bacteria, they may have
stronger bactericidal effects.

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444
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447
448
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450

Dose-dependent effects of antibiotics

451

The dose-dependent effects of three antibiotics on E. coli and K.

pneumoniae were assessed. As shown in Fig. 8, the growth of E. coli
and K. pneumoniae in MHB was continuous in the absence of
antibiotics. As we expected, various concentrations of gentamicin,
cefotaxime, and meropenem inhibited growth. Gentamicin was
the least inhibitory antibiotic for both strains tested. Greater
inhibition was observed with increasing concentrations of
gentamicin (0.01 to 1.0 mg/mL), and complete inhibition was
observed at 1.0 mg/mL, consistent with the MIC value. In both
strains, cefotaxime and meropenem completely inhibited growth
at concentrations of 0.5 and 0.4 mg/mL, respectively. Among the
three tested antibiotics, meropenem showed complete inhibition
of growth at the lowest concentration (0.4 mg/mL). Therefore,
further studies of the synergistic effects of AgNPs and antibiotics
focused on meropenem.

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Evaluation of antibacterial effects of antibiotics with AgNPs

467

The potential additive or synergistic antibacterial effects of

antibiotics and AgNPs were evaluated using the disc diffusion
method. All three antibiotics testedgentamicin (10 mg/mL),
cefotaxime (30 mg/mL), and meropenem (10 mg/mL)showed

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Fig. 8. Effect of antibiotics on cell survival. E. coli and K. pneumoniae cells were incubated with various concentrations of gentamicin, cefotaxime, and meropenem. Bacterial
survival was determined at 4 h by a CFU count assay. The experiment was performed with various controls including a positive control (AgNPs and MHB, without inoculum)
and a negative control (MHB and inoculum, without AgNPs). The results are expressed as the means  SD of three separate experiments, each of which contained three
replicates. Treated groups showed statistically signicant differences from the control group by Students t test (p < 0.05).

Please cite this article in press as: S. Gurunathan, J. Ind. Eng. Chem. (2015), http://dx.doi.org/10.1016/j.jiec.2015.04.005

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S. Gurunathan / Journal of Industrial and Engineering Chemistry xxx (2015) xxxxxx

Fig. 9. Enhancement of antibacterial activity of antibiotics in the presence of AgNPs.

Antibacterial activities were determined by the agar diffusion method. Growth
inhibition was determined by measuring the zone of inhibition after 24 h.
Experiments were performed in triplicate. The percent enhancement of
antibacterial activity was calculated using the formula (BA)/A  100, as
described in the Materials and methods section. The results are expressed as the
means  SD of three separate experiments. Treated groups showed statistically
signicant differences from the control group by Students t test (p < 0.05).

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signicant (p < 0.05) antibacterial effects against both E. coli and

K. pneumoniae (Fig. 9). The activities of all three antibiotics against
both bacterial strains were increased in combination with AgNPs;
no signicant differences were observed between E. coli and
K. pneumoniae, perhaps because both species are gram-negative.
Among the three antibiotics, meropenem showed the largest
increase in activity (75%) when combined with AgNPs, followed by
cefotaxime (63%) and gentamicin (48%). Hwang et al. [51] observed
synergistic activities of silver nanoparticles in the presence of
conventional antibiotics and suggested that bacterial viability was
decreased at lower concentrations of antibiotics. Kora and Rastogi
[30] reported that the combined effects of silver nanoparticles and
antibiotics were more prominent with PVP-capped nanoparticles
than with citrate- or SDS-capped nanoparticles in both grampositive and gram-negative bacteria.

487

Synergistic antibacterial effect of antibiotics and AgNPs

488
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490

Next, we explored the possibility of using AgNPs as an antibiotic

adjuvant. Morones-Ramirez et al. [52] reported that Ag+ could
enhance the bactericidal effect of antibiotics owing to the

generation of ROS [52,53]. To analyze the synergistic effect of

antibiotics and AgNPs, we selected a sublethal dose of meropenem
and examined the antibacterial activity against both E. coli and K.
pneumoniae. Exponentially growing bacteria were incubated with
a sublethal concentration of antibiotic or AgNPs or a combination
of both. Bacteria were harvested at different time points to
determine the number of CFUs. The surviving colonies were
enumerated after 24 h. The CFU assays showed that a sublethal
concentration of antibiotic or AgNPs alone had no signicant
killing effect on either tested strain. However, the combination of
sublethal concentrations of meropenem and AgNPs signicantly
reduced the viability of E. coli and K. pneumoniae cells by more than
75% (Fig. 10). Comparing the antibacterial effects of meropenem,
AgNPs, and their combination, the augmented antibacterial effect
of the antibiotic in combination with AgNPs is noticeable.

491
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500
501
502
503
504
505

Synergistic effect of antibiotics and AgNPs on ROS generation

506

Silver and antibiotics are known to enhance the production of

ROS by increasing the permeability of bacterial membranes;
increased production of ROS induces cell death and may be a
common mechanism of bactericidal antibiotics [5358]. Recently,
several studies have shown that lethal doses of bactericidal
antibiotics promote the formation of highly detrimental ROS
[53,57]. The mechanisms of cell death, oxidative stress, and ROS
formation are some of the key mechanisms of cellular defense
after particle uptake. Nanoparticles could hasten intracellular
oxidative stress by disturbing the equilibrium between oxidant
and antioxidant processes [45]. The generation of ROS is a key
mechanism of toxicity. Therefore, we investigated the effect of
sublethal concentrations of antibiotics, AgNPs, or combinations of
antibiotics and AgNPs in E. coli and K. pneumoniae. Cells were
treated with an antibiotic, AgNPs, or both, and ROS levels were
measured. The ROS levels in cells treated with an antibiotic or
AgNPs were lower than those in cells treated with a combination of
an antibiotic and AgNPs (Fig. 11). Elevated ROS and free radical
levels are candidate mediators of cell death. The production of ROS
could be caused by impeded electron transport along the
respiratory chain in the damaged plasma membrane [50]. The
underlying mechanisms of ROS production could be the reason for
cell death. The mechanism underlying the bactericidal effect of
AgNPs against bacteria remains unclear and further studies are
required. Our results provide some evidence that cell death due to
ROS generation is one of the mechanisms of AgNPs, antibiotics, or a
combination of both antibiotics and AgNPs. On the other hand,
AgNPs may attach to the surface of the cell membrane and disturb

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Fig. 10. AgNPs enhance the bactericidal effect of antibiotics. Bacterial strains were treated with a sublethal concentration of meropenem or AgNPs or a combination of AgNPs
and meropenem for 4 h. Bacterial survival was determined at 4 h by CFU assay. The experiment was performed with various controls including a positive control (AgNPs and
MHB, without inoculum) and a negative control (MHB and inoculum, without AgNPs). The results are expressed as the means  SD of three separate experiments, each of which
contained three replicates. Treated groups showed statistically signicant differences from the control group by Students t test (p < 0.05).

Please cite this article in press as: S. Gurunathan, J. Ind. Eng. Chem. (2015), http://dx.doi.org/10.1016/j.jiec.2015.04.005

G Model

JIEC 2472 110

S. Gurunathan / Journal of Industrial and Engineering Chemistry xxx (2015) xxxxxx

that AgNPs synthesized using leaf extract can be used as an

antibiotic adjuvant for the treatment of various infectious diseases
caused by gram-negative bacteria. Finally, these results suggest a
possible mechanism for the synergistic effects of antibiotics and
AgNPs. Thus, our ndings support the claim that AgNPs have
considerable antibacterial activity that can be used to enhance the
action of existing antibiotics against gram-negative bacteria.
Uncited reference
[74].
Acknowledgements

Fig. 11. Synergistic effect of antibiotics and AgNPs on ROS generation. E. coli and K.
pneumoniae were treated with a sublethal concentration of meropenem or AgNPs or
a combination of AgNPs and meropenem for 24 h. ROS generation was measured by
an XTT assay. The results are expressed as the means  SD of three separate
experiments, each of which contained three replicates. Treated groups showed
statistically signicant differences from the control group by Students t test (p < 0.05).

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membrane functions such as permeability and respiration, and it

has been suggested that the binding of the particles to the bacteria
depends on the surface area available for interaction [59]. Our
study supports the idea that smaller nanoparticles may be more
effective in inducing cell death than larger particles.
Kvitek et al. [60] also suggested that AgNPs may attach to the
surface of the cell membrane, disturbing permeability and
respiration, and that smaller AgNPs having a larger surface area
available for interaction would have greater bactericidal effects
than larger AgNPs. Silver binds to the bacterial cell wall and cell
membrane and inhibits the respiration process [61]. In the case of
E. coli, silver acts by inhibiting the uptake of phosphate and by
releasing phosphate, mannitol, succinate, proline, and glutamine
from the cells [62]. Recently, Balaji Raja and Singh [63] reported
that the antibacterial activity of cephalexin against E. coli was
increased in the presence of AgNPs. Furthermore, the combination
of silver nanoparticles and graphene nanocomposites exhibited
enhanced antibacterial activity against E. coli and S. aureus
compared with individual silver nanoparticles, reduced graphene
oxide nanosheets, or their nanocomposites [63]. Previously,
several studies demonstrated that improvement of antibacterial
activity of AgNPs through silver ion release using nanocomposites
[6473]. Altogether, these results consistent with previous studies
and also indicate that the generation of ROS might be a crucial
mechanism of cell death mediated by AgNPs or a combination of
antibiotics and AgNPs.

561

Conclusion

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In this work, we describe a green approach for the synthesis of

silver nanoparticles using leaf extract of T. angustifolia. The
prepared AgNPs were characterized using various analytical
techniques. The synthesized AgNPs were uniform and had an
average size of 8 nm. We demonstrated that the antibacterial
activity of AgNPs and antibiotics against E. coli and K. pneumoniae
was dose dependent. Furthermore, the antibacterial activity of
gentamicin, cefotaxime, and meropenem against the tested strains
was increased in the presence of AgNPs. Interestingly, the
combination of sublethal concentrations of an antibiotic and
AgNPs signicantly decreased cell viability and increased ROS
production. This study provides evidence of the antibacterial
effects of AgNPs and their synergistic activity with antibiotics
against gram-negative bacteria. Additionally, these results suggest

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Q5583

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This paper was supported by the SMART-Research Professor 586

Program of Konkuk University. Dr. Sangiliyandi Gurunathan was Q6587
supported by a Konkuk University KU-Full time Professorship.
Q7588
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