Microbial Pathogenesis 101 (2016) 1e11

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Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Green synthesis of silver, gold and silver/gold bimetallic nanoparticles

using the Gloriosa superba leaf extract and their antibacterial and
antibiofilm activities
Kasi Gopinath a, Shanmugasundaram Kumaraguru b, Kasi Bhakyaraj b,
Subramanian Mohan b, Kunga Sukumaran Venkatesh c, Masanam Esakkirajan d,
Periyannan Kaleeswarran a, Naiyf S. Alharbi e, Shine Kadaikunnan e,
Marimuthu Govindarajan f, Giovanni Benelli g, Ayyakannu Arumugam h, *
a
Department of Nanoscience and Technology, Alagappa University, Karaikudi 630 003, Tamil Nadu, India
b
EMFT Division, CSIR-Central Electrochemical Research Institute, Karaikudi 630 003, Tamil Nadu, India
c
Multifunctional Materials Laboratory, Department of Physics, International Research Centre, Kalasalingam University, Krishnankoil 626 126, Tamil Nadu,
India
d
Department of Animal Health and Management, Alagappa University, Karaikudi 630 003, Tamil Nadu, India
e
Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
f
Unit of Vector Control, Phytochemistry and Nanotechnology, Department of Zoology, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India
g
Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, Pisa 56124, Italy
h
Department of Botany, Alagappa University, Karaikudi 630 003, Tamil Nadu, India

a r t i c l e i n f o a b s t r a c t

Article history: The green fabrication of metal nanoparticles using botanical extracts is gaining increasing research
Received 5 July 2016 attention in nanotechnology, since it does not require high energy inputs or the production of highly
Received in revised form toxic chemical byproducts. Here, silver (Ag), gold (Au) and their bimetallic (Ag/Au) nanoparticles (NPs)
1 October 2016
were green synthesized using the Gloriosa superba aqueous leaf extract. Metal NPs were studied by
Accepted 17 October 2016
Available online 17 October 2016
spectroscopic (UVevisible spectroscopy, fluorescence spectroscopy, FT-IR spectroscopy, XRD and EDX)
and microscopic (AFM and TEM) analysis. AFM and TEM showed that Ag and Au NPs had triangular and
spherical morphologies, with an average size of 20 nm. Bimetallic Ag/Au NPs showed spherical shapes
Keywords:
Biofabrication
with an average size of 10 nm. Ag and Ag/Au bimetallic NPs showed high antibacterial and antibiofilm
Biosafety activities towards Gram-positive and Gram-negative bacteria. Overall, the proposed synthesis route of
Gloriosa superba Ag, Au and Ag/Au bimetallic NPs can be exploited by the pharmaceutical industry to develop drugs
Nanotechnology effective in the fight against microbic infections.
Surface plasmon resonance © 2016 Elsevier Ltd. All rights reserved.

1. Introduction antibacterial [13,14] antifungal [15], antibiofilm [16,17], entomo-

The green synthesis, also known as phytosynthesis, of noble
metal nanoparticles (NPs) is one of the emerging fields in nano-
science and nanotechnology. The nanoparticle size and shapes
strongly impact the physical, chemical, electrical and optical
properties of nanomaterials. Nowadays, silver (Ag) and gold (Au)
NPs are used in a wide range of biomedical [1,2], drug delivery [3,4],
catalytic [5,6], agriculture [7,8] antioxidant [9,10], anticancer [11,12]
* Corresponding author.
E-mail address: ayyakannuarumugam@gmail.com (A. Arumugam).
logical and parasitological applications [18,19]. Recently, botanical-
based synthesis of Ag and Au NPs have gained a growing attention
due to its low cost, simplicity and environmentally benign syn-
thesis protocols [20e26]. For example, Ag, Au and Ag/Au bimetallic
NPs have been synthesized using plant products from Anacardium
occidentale [27], Piper pedicellatum [28] Punica granatum [29] and
Brassica oleracea [30].
The biofilm is a microbial population enclosed in a matrix. It
grows in three steps, namely (i) initial adhesion, (ii) proliferation
and (iii) detachment. Bacterial cells bind together by extracellular
polymeric substances and connected to a surface (substrate),
involving in each cell-surface and cell-cell interactions as a part of

http://dx.doi.org/10.1016/j.micpath.2016.10.011
0882-4010/© 2016 Elsevier Ltd. All rights reserved.
2 K. Gopinath et al. / Microbial Pathogenesis 101 (2016) 1e11

the developmental process [31,32]. Biofilms show thousand times visually detected.
of resistance towards toxicants if compared to planktonic cells [33].
They can cause serious problems in the environment and leads to
2.4. Characterization of Ag, Au and Ag/Au bimetallic nanoparticles
infections both humans and animals [34e37]. Recently, metal and
metal oxide NPs have been used to inhibit the microbial growth and
We studied the UVevis spectra of the biosynthesized Ag, Au and
forestall the biofilm formation [17,38,39]. The green synthesis of
Ag/Au bimetallic NPs in the 200e850-nm wavelength range using a
protein-stabilized Ag NPs, coated on the surface of the poly-
Shimadzu spectrophotometer (Model UV-1800, Japan) set at a
caprolactam (polymer) evidences the high antibiofilm activity
resolution of 1 nm. The fluorescence study was conducted in the
against bacterial and fungal pathogens [40]. Biosynthesized Ag NPs
range of 200e700 nm with an Elico SL 174 spectrofluorometer. We
showed high activity against the primary biofilm formation of
performed a FTIR analysis (Thermo Nicolet 380, USA) in the 400 to
Pseudomonas aeruginosa and Staphylococcus aureus [41]. Also,
4000 cm1 range. The XRD pattern was captured using Cu Ka ra-
sixteen different marine biofilm bacterial isolates have been
diation (l ¼ 1.54060 Å) with a nickel monochromator in the 2q
investigated for the antibiofilm activity, treating them with
10
e80
range with a PANanalytical X-PERT PRO diffractometer.
different concentrations of biosynthesized Ag NPs. It has been
The mean crystallite size of the biosynthesized silver, gold and
noted that 50 mg/mL of Ag NPs significantly exhibited the preven-
silver/gold bimetallic nanoparticles was calculated using Scherrer's
tion of biofilm with some variations among the tested bacteria [16].
formula [D ¼ 0.9l/bcos q]. We conducted EDX analysis (FEI Quanta
Gloriosa superba L., (Colchicaceae) is a climbing herb native of
250, Czech Republic) for thin film samples prepared using the sil-
South Africa. It is a national flower of Zimbabwe and state flower of
ver, gold and silver/gold bimetallic nanoparticles via a method of
Tamil Nadu [42]. G. superba is a tuberous plant with plow shaped
spin coating (1500 rpm) on a 1x1-cm aluminum foil, by dropping
cylindrical tubers. It is commercially cultivated in Tamil Nadu, and
100 mL of each sample on the foil and leaving it for 30 min at room
particularly in Karur and Tirupur districts. It has been used in ail-
temperature to dry. For AFM analysis, we prepared the samples on a
ments of arthritis, gout, rheumatism, inflammation, ulcers,
1x1-cm glass slide by dropping 100 mL of each sample on the slide
bleeding piles, skin diseases, leprosy and snakebites [43,44]. The
and leaving it for 30 min to dry. Subsequently, we scanned the
leaf extract contains superbine, colchicine, gloriosine, gloriosol,
slides with AFM (APE Research-model no. A100-SGS). The AFM
phytosterils and stigmasterin [44]. Recently, Ashok Kumar et al. [5],
assessment was conducted in ambient temperature in a non-
focused on green synthesis of AgNPs using G. superba leaf extract
contact mode using silicon nitrite tips with different resonance
and the related catalytic activity.
frequencies. The morphology of the biosynthesized silver, gold and
In this research, silver (Ag), gold (Au) and their bimetallic (Ag/
silver/gold bimetallic nanoparticles was evaluated with the use of
Au) nanoparticles (NPs) were green synthesized using the
TEM. We prepared the samples for TEM analysis by drop coating
G. superba aqueous leaf extract. The synthesized metal NPs were
the nanoparticle solutions on carbon-coated copper grids and
characterized by spectroscopic (UVevisible spectroscopy, fluores-
leaving them to dry at room temperature. TEM analysis was con-
cence spectroscopy, FT-IR spectroscopy, XRD, and EDX) and
ducted on a Tecnai F20 model set at an accelerating voltage of
microscopic (AFM and TEM) analyses. Furthermore, Ag and Ag/Au
bimetallic NPs were studied for their high antibacterial and anti-
biofilm activities towards Gram-positive and Gram-negative
bacteria.

2. Materials and methods

2.1. Collection of plant material

We gathered the leaves of G. superba plants from the Endan-

gered Medicinal Plants Conservation Centre, Science Campus, Ala-
gappa University, Karaikudi, Tamil Nadu, India.

2.2. Chemicals

The chemicals used for the synthesis were of analytical grade.

We purchased silver nitrate (AgNO3) and chloroauric acid
(HAuCl4.3H2O) from Merck (India) and Sigma-Aldrich Chemicals
(USA), respectively. Moreover, we acquired microbiological media
were from HiMedia Laboratories, Pvt. Ltd, India.

2.3. Synthesis of Ag, Au and Ag/Au bimetallic nanoparticles

We cutted the leaves of G. superba into fine pieces, then we

added 10 g of them to 100 mL of double-distilled water, before
heating the mixture at 50e60
C for 5 min. We filtered the extract
through Whatman n. 1 filter paper and we collected the filtrate in a
250-mL Erlenmeyer flask, before storing it at room temperature for
further use. 5 mL of the G. superba leaf extract was mixed with
100 mL of each of the following solutions: Silver nitrate 1 mM (pH
4.35), Chloroauric acid 1 mM (pH 2.26), and silver nitrate/chlor-
oauric acid (1:1) (pH 5.2), at room temperature. After 10 min, the Fig. 1. Ag, Au and Ag/Au bimetallic nanoparticles (NPs) phytosynthesized using the
reduction of Ag, Au, and Ag/Au bimetallic nanoparticles was Gloriosa superba leaf extract.
 K. Gopinath et al. / Microbial Pathogenesis 101 (2016) 1e11
200 kV.
2.5. Antibacterial activity of Ag, Au and Ag/Au bimetallic
nanoparticles
We evaluated the antibacterial activity of the synthesized Ag, Au
and Ag/Au bimetallic NPs with the disc diffusion method for both
Gram-positive (Bacillus subtilis ATCC 6633) and Gram-negative
bacteria (Escherichia coli MTCC 40). We cultured the bacteria in a
37
C nutrient broth until their population reached 1.5  108 CFU/
mL. Added 20 mL of molten nutrient agar into the Petri dishes and
left it to cool. After swabbing the bacterial suspension to the me-
dium, sterile forceps were used to add 10, 30 and 50 mL of silver,
gold and silver/gold bimetallic nanoparticles. We used 50 mL of
G. superba leaf extract as a negative and a 30 mg ampicillin disc as a
positive control. The plates were incubated at 37
C for 24 h. Each
experiment was conducted in three repetitions, where we
measured each disc's inhibition zone (mm).
2.6. Antibiofilm activity of Ag, Au and Ag/Au bimetallic
nanoparticles
S. aureus (MTCC 96), S. pneumoniae (MTCC 1936), K. pneumoniae
(MTCC 432) and E. coli (MTCC 40) were cultured at 37
C in Luria
Bertani (LB) broth. For the biofilm formation experiments, the
sterile glass slides having the dimension of 1  1 cm were kept in
the 24-well polystyrene plates containing (total volume - 2.5 mL)
Fig. 2. UVevisible absorbance spectra of metal nanoparticles (NPs) phytosynthesized using the Gloriosa superba leaf extract. Surface plasmon resonance peak are at (a) - 425 nm for
Ag NPs, (b) - 538 nm for Au NPs and (c) - 559 nm Ag/Au bimetallic NPs.
3
2 mL of sterilized LB broth, 250 mL of metal NPs and 250 mL of test
biofilm bacterial strains 1.5  108 CFU/mL, incubated at 37
C for
24 h. The plant leaf extract was tested as negative control. After 24 h
of incubation, glass slides were removed and rinsed three times
with phosphate buffered saline (PBS) solution (25 mL) and then
cells were enumerated by 10 mg/mL of propidium iodide and
staining was continued to 1 min. Therefore, the slides were
examined by confocal laser scanning microscopy (CLSM 710; Carl
Zeiss, Germany).
2.7. Statistical analysis
All the data were expressed as mean ± SE. Data normal distri-
bution was confirmed, then we carried out the analysis of variance
(two-way ANOVA) followed by Tukey's HSD test (P  0.05). All
statistical analyses were carried out using the SPSS software.
3. Results and discussion
During our experiments, we observed the rapid reduction of
silver, gold and silver/gold bimetallic nanoparticles within 10 min
from the addition of silver nitrate, chloroauric acid, and silver ni-
trate/chloroauric acid (1:1) solutions to the leaf extract of
G. superba. The initially uncolored solution was promptly colored
brown, yellow to dark red, and yellowish white to reddish brown,
indicating the swift formation of the silver, gold and silver/gold
bimetallic nanoparticles, respectively (Fig. 1).
4 K. Gopinath et al. / Microbial Pathogenesis 101 (2016) 1e11

3.1. UVevisible spectroscopy and fluorescence spectroscopy 571 nm, which were probably linked to the formation of bimetallic
NPs. The curve fitting clearly illustrates the embedded emission
The plant leaf extract, phytosynthesized Ag, Au and Ag/Au peaks [45,46].
bimetallic NPs solution were subjected to UVevisible spectroscopy
during the rapid reduction process, in order to shed light the
mechanism of Ag, Au and Ag/Au bimetallic NPs formation. The 3.2. Fourier transform infrared spectroscopy
recorded spectra at different time intervals such as 0, 1, 2, 3, 4, 5 and
10 min are shown in Fig. 2. The spectra recorded for the leaf extract We employed a FT-IR spectroscopy to identify the possible
did not showed the any absorption peak between the region from
200 to 850 nm, while the recorded spectra during the rapid
reduction process at different time intervals showed absorption
peaks at 425, 538 and 559 nm, which corresponds to the wave-
length of the surface plasmon resonance (SPR) of Ag, Au and Ag/Au
bimetallic NPs. Our results are in agreement with recent previous
reports [28,29]. The Ag/Au bimetallic NPs showed an absorption
peak at 559 nm, which is a good agreement with the bimetallic NPs
(Ag:Au) 1:1 ratio onwards [30]. The fluorescence properties of the
plant leaf extract, phytosynthesized Ag, Au and Ag/Au bimetallic
NPs were recorded in water and emission peaks were observed at
471, 530, 540 and 530, 571 nm (Fig. 3) respectively. G. superba leaf
extract showed an emission peak at lem ¼ 471 nm, which may be
due to the phytocompounds colchicine, gloriosine and gloriosol
characterizing the leaf extract [44]. However, the emission spectra
of green fabricated Ag NPs consisted in two mean emission peaks.
The longer emission peak at 471 nm was attributed to the leaf
extract and another shorter emission peak at 530 nm, indicates the
formation of Ag NPs. A shorter emission peak at 540 nm, confirms
the formation of Au NPs. In addition, the synthesized Ag/Au
Fig. 4. FT-IR spectra of Gloriosa superba leaf extract and phytosynthesized Ag, Au and
bimetallic NPs showed two shorter emission peaks at 530 and Ag/Au bimetallic nanoparticles.

Fig. 3. Fluorescence spectra: (a) emission peak characterizing the Gloriosa superba leaf extract at 471 nm, (b) Ag nanoparticles (NPs), peak at 530 nm, (c) Au NPs, peak at 540 nm,
and (d) Ag/Au bimetallic NPs, peaks at 530 and 571 nm.
 K. Gopinath et al. / Microbial Pathogenesis 101 (2016) 1e11 5

biomolecules that were responsible for the reduction of the Agþ

and Auþ ions. We noted strong IR bands at 3418, 2922, 1633, 1384,
1259, 1070 and 666 cm1 (Fig. 4). The bands appearing at 3418 and
2922 cm1 correspond to the stretching of primary amines (NeH)
and aliphatic (CeH) respectively. The 1633 and 1384 cm1 bands
correspond to the stretching of C]C and NO2 respectively. The
1259 and 1070 cm1 bands appear due to the stretching of Ce
OeCeOeC. The 666 cm1 low band can be attributed to the
stretching of CeCl. Consequently, primary components of the
G. superba leaf extract, such as colchicine, superbine, gloriosol,
gloriosine, stigmasterin, and phytosterils, are responsible for the
observed reduction, and for the completion of the biosynthetic
process of silver, gold, and silver/gold bimetallic nanoparticles [44].
After the synthesis of the nanoparticles, the band in the 1259 cm1
spectrum disappeared, indicating that the glycosides and the water
soluble tannins that were present in the leaf extract facilitated the
reduction of the metal ions to metal nanoparticles [27,47].

3.3. X-ray diffraction and energy dispersive X-ray spectroscopy

The powder X-ray diffraction patterns showed the nano-

crystalline nature of silver, gold and silver/gold bimetallic nano-
particles as reported in Fig. 5. The noted diffraction peaks and the
appropriate planes such as (111), (200), (220) and (311) corre-
sponded to the face-centered cubic silver, gold and silver/gold
bimetallic nanoparticles, respectively. The characteristic peak
values were conform to the standard data (JCPDS card no. 89-3722,
04-0784 for silver and gold respectively). The well-resolved and
strong pattern of the XRD distinctly showed that the silver and gold
nanoparticles that are formed due to the Agþ and Auþ ion reduction
by the leaf extract of G. superba, are crystalline in nature. The silver/
gold bimetallic nanoparticles were not different over the silver and
gold nanoparticles, this can be explained by their similar crystal
lattice structure. We used Scherrer's equation to assess the mean
crystallite size and calculated it at 26.53, 21.87 and 14.25 nm for
silver, gold and silver/gold bimetallic nanoparticles respectively.
Finally, we conducted energy dispersive X-ray spectroscopy (EDX)
to illustrate the elemental composition of the metal nanoparticles
that were biosynthesized by the leaf extract of G. superba. The peaks
were noted at 3.0, 2.12 and 3.0, 2.12 keV for silver, gold and silver/
gold bimetallic nanoparticles, as shown in Fig. 6. We observed a

Fig. 6. EDX spectra of phytosynthesized (a) Ag nanoparticles (NPs), (b) Au NPs and (c)
Ag/Au bimetallic NPs.

significant peak corresponding to Al in all samples, which is

attributed to the aluminum foil substrate.
Fig. 5. XRD pattern of phytosynthesized Ag, Au and Ag/Au bimetallic nanoparticles
(NPs).
6 K. Gopinath et al. / Microbial Pathogenesis 101 (2016) 1e11
3.4. Atomic force microscopy and transmission electron microscopy
We carried out AFM analysis to examine the surface topology of
the biosynthesized silver, gold and silver/gold bimetallic nano-
particles (Fig. 7). It is evident from the micrographs that the syn-
thesized silver and gold nanoparticles are triangular and spherical
in shape, with their measured size ranging from 20 to 50 nm.
Moreover, the silver/gold bimetallic nanoparticles were spherical,
with size ranging from 10 to 20 nm. Images from TEM, shown in
Fig. 8a, b, d and e, provide further evidence of the predominantly
triangular and spherical morphology of the silver and gold nano-
particles, as well as of their size in the range of 20e50 nm. The same
was proved for silver/gold bimetallic nanoparticles, as their
spherical morphology and their mean size of 10 nm were also
confirmed (Fig. 8g and h). The selected area electron diffraction
(SAED) pattern (Fig. 8c, f and i) of silver, gold and silver/gold
bimetallic nanoparticles confirmed the characteristic ring pattern
of face-centered cubic (fcc), which added further evidence to the
notion that the silver, gold, and silver/gold nanoparticles where
highly crystalline in nature.
3.5. Antibacterial activity
The antibacterial activity of phytosynthesized Ag, Au and Ag/Au
bimetallic NPs was evaluated against Gram-positive and Gram-
negative bacterial pathogens (Fig. 9). 30 mL of Ag NPs showed a
Fig. 7. AFM images of phytosynthesized metal nanoparticles (NPs): (a-b) 2D and 3D image of Ag NPs, (c-d) 2D and 3D image of Au NPs, (e-f) 2D and 3D image of Ag/Au bimetallic
NPs.
significant effect in E. coli with the zone of inhibition
(7.66 ± 0.33 mm). Au NPs did not showed toxicity towards the
tested bacterial strains. When 50 mL of Ag/Au bimetallic NPs were
tested, we observed a significant zone of inhibition on B. subtilis
(6.33 ± 0.33 mm) as well as a modulated effect on E. coli
(5.33 ± 0.33 mm). This efficacy of antibacterial activity of Ag/Au
bimetallic NPs could be due to the synergistic effect of bimetallic
formation (Table 1).
The exact mechanism behind the metal nanoparticles' antibac-
terial activity is poorly understood [48]. However, the ability of
silver nanoparticles to form electrostatic bonds with the bacterial
membrane, where the positively charged particles and the nega-
tively charged membrane interact, seems to play a crucial role; the
interaction disrupts the bacterial membrane's integrity resulting in
cell death -hence the bactericidal action. Silver nanoparticles bind
with both the cell membrane and the mesosomes, leading to a
disruption of mesosomal function, which leads to an increase in the
production of reactive oxygen species, causing the cell to die [48].
Furthermore, gold nanoparticles may also bind to the bacterial
membrane but, due to their low toxicity, they may not cause
cellular death. Ag/Au bimetallic NPs have more significant effect
and showed higher inhibition zone formation than the Ag NPs, and
this may be due to the synergistic effect arising from the Ag/Au
bimetallic NPs. The Ag/Au bimetallic NPs consists different prop-
erties mainly the antibacterial activity of Ag/Au bimetallic NPs was
interfere with the bacterial inter-cellular cell signaling and its
 K. Gopinath et al. / Microbial Pathogenesis 101 (2016) 1e11 7

Fig. 8. (a, b) TEM of Ag nanoparticles (NPs), (c) SAED pattern of Ag NPs; (d, e) TEM of Au NPs, (f) SAED pattern of Au NPs; (g, h) TEM of Ag/Au NPs and (i) SAED pattern of Ag/Au
bimetallic NPs.

malfunction of cell metabolisms. Secondly, the random effect of
mutation, which occurs due to the loss of bacterial cell viability. It
has high synergistic strength when compared to the Ag and Au NPs.
3.6. Antibiofilm activity of the nanoparticles
After 24 h incubation of the biofilm formation, the CLSM images
indicated weak adherence and disintegrated biofilm surface to-
pology of all the tested bacterial strains, i.e. S. aureus, S. pneumoniae,
K. pneumoniae and E. coli (Fig. 10). In the control, it was observed
that the dense as well as strongly adhesive biofilm formation of
human pathogenic bacterial strains on the glass substrates. The
antibiofilm activity of Ag/Au bimetallic NPs showed a more sig-
nificant inhibition of biofilm growth thickness at 8, 11, 9 and 14 mm
for S. aureus, S. pneumoniae, K. pneumoniae and E. coli respectively,
which is due to the synergistic effect of bimetallic NPs. Subse-
quently, Ag NPs exhibited a better effect of inhibited biofilm growth
at 15, 18, 16 and 19 mm for S. aureus, S. pneumoniae, K. pneumoniae
and E. coli respectively. Au NPs did not show inhibition of biofilm
formation, which is similar to the control (Table 2). It has been
shown that a mechanistic interaction exists between silver nano-
particles and the bacterial membrane, which results in the bacterial
8 K. Gopinath et al. / Microbial Pathogenesis 101 (2016) 1e11

Fig. 9. The antibacterial activity of phytosynthesized Ag nanoparticles (NPs) (top), Au NPs (centre) and Ag/Au bimetallic NPs (bottom) against Bacillus subtilis (left) and Escherichia
coli (right).

cell's death. Due to the electrostatic attraction of the silver nano-
particles, the bacterial cell membrane suffers damage and pits are
formed on its surface, resulting in a series of structural changes that
affect cell respiration [49]. Moreover, silver nanoparticles interact
with thiol groups that are present in proteins, resulting in inhibi-
tion of bacterial protein synthesis and replication of the DNA [50].
In a similar manner, oxygen is associated with Ag and reacts with
the sulfhydryl (-S-H) groups on the cell wall resulting in the
removal of hydrogen ions (in the form of water). This causes the
sulfur atoms to form an R-S-S-R bond, which blocks respiration,
ultimately leading to bacterial cell death [51]. Ag NPs preferably
attack the respiratory chain and cell division, ultimately leading to
cell death [52]. Similarly, citrate capped Ag NPs and silver ions were
tested in antibacterial and antibiofilm activity of P. aeruginosa PA01.
The better activity was observed for the concentration of 10 mg/L
[38]. Previously, it has been reported that Ag NPs inhibited the
natural marine biofilm bacterial population and reduction of bio-
film formation was concentration dependent i.e. from 200 to
2000 mg/L [53].
4. Conclusions
The green fabrication of nanoparticles using botanical extracts is
gaining increasing research attention in nanotechnology and
biomedical applications, since it does not require high energy in-
puts or the production of highly toxic chemical byproducts [54].
Our research pointed out that silver (Ag), gold (Au) and their
bimetallic (Ag/Au) nanoparticles (NPs) can be easily fabricated
 K. Gopinath et al. / Microbial Pathogenesis 101 (2016) 1e11 9

Table 1 Table 2
Antibacterial activity of phytosynthesized Ag, Au and Ag/Au bimetallic nanoparticles Phytosynthesized Ag, Au and Ag/Au bimetallic nanoparticles (NPs): reduction of
(NPs). biofilm thickness in different bacteria.

Treatment Zone of inhibition (mm) Bacteria Thickness of biofilm in (mm)

B. subtilis E. coli Control Ag NPs Au NPs Ag/Au bimetallic NPs

** *
Leaf extract 0.00 ± 0.00 0.00 ± 0.00 S. aureus 30 15 29 8***
10 mL Ag NPs 1.33 ± 0.33d 5.33 ± 0.33c S. pneumoniae 32 18** 30* 11***
30 mL Ag NPs 2.33 ± 0.33cd 7.66 ± 0.33b K. pneumoniae 35 16** 28* 9***
50 mL Ag NPs 3.00 ± 0.33c 7.33 ± 0.33b E. coli 34 19** 31* 14***
Ampicillin 0.00 ± 0.00 10.33 ± 0.33a
***Significant.
10 mL Au NPs 0.00 ± 0.00 0.00 ± 0.00
**Modulated.
30 mL Au NPs 0.00 ± 0.00 0.00 ± 0.00
*Not significant.
50 mL Au NPs 0.00 ± 0.00 0.00 ± 0.00
Ampicillin 0.00 ± 0.00 11.33 ± 0.33a
10 mL Ag/Au bimetallic NPs 3.33 ± 0.33c 3.33 ± 0.33c
30 mL Ag/Au bimetallic NPs 5.00 ± 0.00b 4.33 ± 0.33bc using the G. superba aqueous leaf extract. AFM and TEM showed
50 mL Ag/Au bimetallic NPs 6.33 ± 0.33a 5.33 ± 0.33b that Ag and Au NPs had triangular and spherical morphologies,
Ampicillin 0.00 ± 0.00 10.33 ± 0.33a with an average size of 20 nm. Bimetallic Ag/Au NPs showed
Within a column, means followed by the same letter are significantly different spherical shapes with an average size of 10 nm. The Ag and Ag/Au
(ANOVA, Tukey's HSD test, P  0.05). bimetallic NPs showed high antibacterial and antibiofilm activities

Fig. 10. The antibiofilm activity of phytosynthesized Ag, Au and Ag/Au bimetallic nanoparticles (NPs) against Gram-positive (S. aureus and S. pneumoniae) and Gram-negative
bacteria (K. pneumoniae and E. coli).
10 K. Gopinath et al. / Microbial Pathogenesis 101 (2016) 1e11
towards Gram-positive and Gram-negative bacteria. Overall, the
proposed synthesis route of Ag, Au and Ag/Au bimetallic NPs can be
exploited by the pharmaceutical industry in order to develop drugs
effective in the fight against microbic infections.
Acknowledgements
The authors extend their sincere appreciations to the Deanship
of Scientific Research at King Saud University for its funding this
Prolific Research Group (PRG-1437-36). The authors are gratefully
thank School of Physics, Alagappa University for extending the XRD
facility and also the Department of Industrial Chemistry, Alagappa
University for providing the fluorescence analysis and EDX facil-
ities. We sincerely thank the Department of Animal Health and
Management, Alagappa University, for providing the CLSM facility.
We thank Swami Shukadevananda, secretary, RKM Vivekananda,
College, Chennai, for extending the UVevisible spectroscopy facility
and kindly providing bacterial strains. G. Benelli is sponsored by
PROAPI (PRAF 2015) and University of Pisa, Department of Agri-
culture, Food and Environment (Grant ID: COFIN2015_22). Funders
had no role in the study design, data collection and analysis, deci-
sion to publish, or preparation of the manuscript.
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