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 Nanotechnology

Nanotechnology 25 (2014) 325101 (16pp) doi:10.1088/0957-4484/25/32/325101

Green synthesis of Kocuran-functionalized

silver glyconanoparticles for use as
antibiofilm coatings on silicone urethral
catheters
C Ganesh Kumar1,2 and Pombala Sujitha1,2
1
Academy of Scientific and Innovative Research, CSIR-Indian Institute of Chemical Technology, Uppal
Road, Hyderabad 500007, India
2
Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Uppal
Road, Hyderabad 500007, India

E-mail: cgkumar@iict.res.in and cgkumar5@gmail.com

Received 7 March 2014, revised 26 May 2014

Accepted for publication 17 June 2014
Published 25 July 2014

Abstract
Microbial infections due to biofilm formation on medical implants are serious complications
arising after surgery which can be prevented by using antimicrobial coatings on biomaterial
surfaces. We developed a simple, rapid and green chemistry approach for synthesis of silver
glyconanoparticles (AgNPs) using Kocuran, an exopolysaccharide produced by Kocuria rosea
strain BS-1. Kocuran-capped AgNPs exhibited a characteristic surface plasmon resonance (SPR)
peak around 435 nm. They were mono-dispersed, spherical with an average particle size of
12 nm. XRD and SAED studies suggested that AgNPs were crystalline in nature. AgNPs had a
zeta potential of −33.9 mV and were anionic charged. They showed colloidal stability at different
pH (6 to 10), temperatures (30 °C to 100 °C), in NaCl, NaNO3 and BSA solutions. Kocuran-
capped AgNPs exhibited effective antimicrobial activity against Staphylococcus aureus and
Escherichia coli and cell death was mainly due to hydroxyl radical induction and depletion of
NADH. They also inhibited the biofilm development by S. aureus and E. coli and confocal
scanning laser microscopic images revealed the damage of intact cell architecture. In vitro
evaluation of Kocuran-capped silver glyconanoparticles on human gingival fibroblasts
demonstrated good cell proliferation as compared to commercial AgNPs suggesting that they are
biocompatible and non-toxic in nature. This is a first report on Kocuran-functionalized AgNPs
exhibiting potential antibacterial and antiadhesive properties for use as antimicrobial coatings
against bacterial adhesion and biofilm formation on silicone urethral catheters.

Keywords: glyconanoparticles, antibiofilm coatings, urethral catheters, Kocuran,

exopolysaccharide
(Some figures may appear in colour only in the online journal)

1. Introduction biofilm formation onto the implant surfaces by opportunistic

Medical devices such as implants, prosthetics, etc are widely
used in surgical cases for the replacement of damaged or lost
tissues and also finds use in critical care for fluid or gas
administration using catheters, respectively. Bacterial con-
tamination during surgery and subsequent adhesion and
microbes cause serious infection and implant failure [1].
Prevention of colonization of these pathogens on medical
implants is one the major medical issues which adds to
financial burden since nosocomial infections represents one of
the most serious complications after surgery or critical care. It
is reported that more than 1.4 million people worldwide suffer

0957-4484/14/325101+16$33.00 1 © 2014 IOP Publishing Ltd Printed in the UK

Nanotechnology 25 (2014) 325101
from complications of hospital-acquired infections leading to
a mortality rate of 10–15% [2, 3]. Staphylococcus aureus
(Gram-positive) and Escherichia coli (Gram-negative) are
major causative agents of hospital-acquired infections, parti-
cularly in immuno-compromised patients, which contribute to
increased rates of implant-related systemic infections and
mortality [3]. A recent study demonstrated that different S.
aureus strains are responsible for catheter-related infections
and among them 82% are methicillin-resistant strains having
numerous genes involved in biofilm formation and bacterial
dispersion [4].
Antimicrobial polymers and coatings containing silver
have gained special attention in recent years due to their long-
term biocidal properties, high temperature stability and low
volatility [5]. Silver is known to exhibit low toxicity towards
mammalian cells [6] and does not easily elicit microbial
resistance [7]. It is reported to cause pits in bacterial cell
walls, resulting in increased permeability and cell death [8]. In
general, the antimicrobial materials and coatings should have
desirable attributes such as potent antibacterial efficacy, low
toxicity, ease of fabrication and environmental safety, to
qualify them as promising candidates for use in various
applications. Hence, there is a significant interest in the
development of silver-based antimicrobial materials and
coatings for application in the health and biomedical devices,
personal hygiene and food industries.
Biodirected synthesis of metal nanoparticles has gained
renewed attention which involves the use of various bioma-
terials from plants, bacteria, fungi and yeasts as reducing and
stabilizing agents due to their biocompatibility, low toxicity
and eco-friendly nature [9, 10]. Among the metal nano-
particles, silver nanoparticles have unique optical, electro-
magnetic and physicochemical properties, which are
dependent on their geometrical properties such as size and
shape and function as bio-nanofactories finding a wide range
of applications as antibacterial agents [11], and in the medical
sector as biodiagnostics, imaging, drug delivery, bio-labeling
and hyperthermia of tumors [12, 13]. The size, shape, stability
and bioactivity are dependent on the capping agent used for
silver nanoparticle synthesis. Silver nanoparticle preparations
using microbes such as bacteria, yeast, fungi, and actinomy-
cetes are well documented [10, 14]. The silver nanoparticle
formation occurs in microorganisms either at intracellular
[15] or extracellular [16] level. Biomolecules such as nitrate
reductase, proteins, amino acids and polysaccharides secreted
by the bacteria act as capping ligands during the silver
nanoparticle synthesis [17]. Some natural products like vita-
min E [18], plant extracts [19], honey [20], sweet sorghum
syrup [21], etc have also been explored for the synthesis of
silver nanoparticles.
Glyconanoparticles prepared with sugars as capping
ligands have gained attention in recent years. Recent studies
have demonstrated the preparation of gold and silver glyco-
nanoparticles functionalized with carbohydrates like glucose
[22], lactose [23], Ley tetrasaccharide [24], starch [25, 26] and
rhamnolipids [27]. These carbohydrates possess many
hydroxyl and carbonyl groups which confer the glyconano-
particles with unique H-bonding capabilities functioning as
2
C G Kumar and P Sujitha
smart nanomaterials for biomedical applications as probes of
carbohydrate–carbohydrate interactions and carbohy-
drate–protein interactions, antiadhesive therapy and biolabels
[28, 29]. However, some reports indicate that it is difficult to
produce polysaccharide-based glyconanoparticles with spe-
cific surface functionalities, which is essential in controlling,
for example, distribution or cell interactions in vivo [30, 31].
Considering the importance of glyconanoparticles from a
biotechnological and biomedical perspective, we report for
the first time a simple, rapid and greener approach for the
synthesis of AgNPs functionalized with Kocuran [32] an
exopolysaccharide (EPS) produced by Kocuria rosea strain
BS-1 with repeating monosaccharide residues of D-glucose,
D-mannose, D-galactose and D-glucuronic acid having an
average molecular mass of 51.2 kDa, and the application of
these Kocuran-capped silver glyconanoparticles (AgNPs) as
antimicrobial and antibiofilm coatings on silicone urethral
catheters.
2. Experimental procedures
2.1. Materials
All chemicals were of analytical grade and procured from
Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
All culture media were purchased from HiMedia Laboratories
Pvt., Mumbai, India. The commercially available citrate-
synthesized silver nanoparticles (10 ± 2 nm) were obtained
from Nanocomposix, San Diego, CA, USA. The fluorescent
reporter dye, 3′-(p-hydroxyphenyl) fluorescein (HPF) and
LIVE/DEAD® BacLight™ Bacterial Viability kit were pro-
cured from Molecular Probes, Eugene, OR, USA. Thermanox
plastic cover slips were obtained from Nalgene Nunc Inter-
national, Rochester, NY, USA.
2.2. Kocuran production from K. rosea strain BS-1
K. rosea strain BS-1, earlier isolated in our laboratory from a
petroleum contaminated soil sample produced an EPS with
biosurfactant property [33]. EPS production from strain BS-1
was achieved in a minimal salts medium containing (per litre):
10 g glucose, 2 g sodium nitrate, 0.5 g NH4SO4, 2.5 g
KH2PO4, 2.0 g K2HPO4, 0.2 g MgSO4 and 1 g NaCl, by
culturing at 35 °C in 1000 ml Erlenmeyer flasks containing
250 ml of minimal salts medium (pH 8) with agitation at
180 rpm for 96 h in an Ecotron shaker (Infors AG, Switzer-
land). The fermented medium was later subjected to cen-
trifugation (Sorvall RC 5C Plus; Kendro Lab Products,
Ashville, NC, USA) at 10 000 g for 20 min to obtain a cell-
free supernatant [32]. Kocuran (EPS) content in the cell-free
supernatant was quantified by phenol sulphuric acid
method [34].
2.3. Preparation and characterization of silver nanoparticles
using Kocuran
Ten milliliter of aqueous silver nitrate (100 mM, Sigma,
USA) was mixed with 90 ml of cell-free supernatant
Nanotechnology 25 (2014) 325101
containing Kocuran suspension (10 mg ml−1), and kept for
continuous stirring at 200 rpm for 15 min at 50 °C. Sample
aliquots (2 ml) were withdrawn at periodic intervals and
monitored for the bioreduction of silver ions. UV–visible
spectra of these sample aliquots were recorded as a function
of reaction time from 300 to 600 nm on a UV–visible double
beam spectrophotometer (Lambda 25, PerkinElmer, Shelton,
CT, USA) at room temperature (28 °C). The nanoparticles
were washed with distilled water, subjected to centrifugation
(Sorvall RC 5C Plus, Kendro Lab Products, Ashville, NC,
USA) at 9700 rpm for 30 min, concentrated and later stored at
4 °C for further use. The synthesized glyconanoparticles were
further characterized using FT-IR, TEM, EDAX, XRD and
zeta potential analysis. The FT-IR spectrum of the AgNPs in
the form of KBr pellets was recorded on Thermo-Nicolet
Nexus 670 spectrometer at a resolution of 4 cm−1 in wave-
number region of 400–4000 cm−1. Morphological analysis of
silver nanoparticle formation was confirmed using transmis-
sion electron microscope (TEM). The powdered AgNPs were
re-dispersed in deionized water by ultrasonication for 5 min
and then subjected to TEM analysis by drying the nano-
particle dispersion on amorphous carbon-coated copper grids
at 37 °C. Kocuran-capped AgNPs were imaged using FEI
Tecnai G2 S-Twin TEM instrument operated at an accel-
erating voltage of 300 kV. The energy dispersive x-ray
(EDAX) spectroscopy was performed on a Scanning Electron
Microscope (Hitachi S-520) equipped with an EDAX detector
(Oxford LINK-ISIS 300) for elemental composition analysis
and EDAX spectrum was measured at 10 kV accelerating
voltage. The x-ray diffraction (XRD) pattern of AgNPs was
recorded employing a XPert PRO P Analytical PW 3040/60
(PANalytical BV, The Netherlands) x-ray diffractometer
operated at a voltage of 40 kV and current of 30mA using
CuKα radiation (λ = 0.154 056 nm) and the scan parameters
set were scan rate 1.2° per minute and scan range
2θ = 10°–80°. The powdered AgNPs dispersed in deionized
water by ultrasonication were also subjected to dynamic light
scattering (DLS) measurements at 25 °C on a Zetasizer Nano
ZS (Malvern Instruments, Worcestershire, UK) instrument
equipped with a He–Ne laser operating at 632.8 nm and a
scattering detector at 173°. The nanoparticle charge quantified
as zeta potential was determined using the same instrument
at 25 °C.
2.4. Stability studies of Kocuran-capped silver
glyconanoparticles
Temperature and pH stability of Kocuran-capped AgNPs was
evaluated in the temperature range of 30 to 100 °C and
pH range of 6 to 10. Further, the stability was assessed in
distilled water, NaCl (different concentrations from 10, 50,
100, 150 and 200 mM), NaNO3 (different concentrations
from 10, 50, 100, 150 and 200 mM), and in BSA solution
(different concentrations ranging from 0.05, 0.1, 0.2, 0.3, 0.4
and 0.5%). In all the cases, the stability of silver glycona-
noparticles was monitored by measuring the absorbance
changes using UV-visible absorption spectroscopy. The sta-
bility of Kocuran-capped AgNPs was monitored by storage at
3
C G Kumar and P Sujitha
room temperature for 60 days; the stored glyconanoparticles
were redispersed in water by sonication to regenerate a sol
followed by UV-visible spectroscopic analysis on differ-
ent days.
2.5. Antimicrobial activity of Kocuran-capped silver
glyconanoparticles
Antimicrobial activity of Kocuran-capped AgNPs in com-
parison with commercially available citrate-synthesized silver
nanoparticles (10 ± 2 nm) was determined using the microtiter
broth dilution method [16]. The pathogenic bacterial strains
used were Bacillus subtilis MTCC 121, S. aureus MLS 16
MTCC 2940, S. aureus MTCC 96, Micrococcus luteus
MTCC 2470, E. coli MTCC 739, Klebsiella planticola
MTCC 530 and Pseudomonas aeruginosa MTCC 2453,
along with drug resistant bacterial strains such as E. coli
ATCC 35218, S. aureus subsp. aureus ATCC 29213 and
Enterococcus faecalis ATCC 29212. The different bacterial
strains (107 cfu ml−1 cells) were inoculated in 100 μl of
Muller–Hinton broth along with different concentrations of
silver glyconanoparticles. After 24 h incubation, 40 μl of p-
iodonitrotetrazolium (INT, Sigma) dye (0.02%, 20 mg INT
dissolved in 100 ml of 40% dimethylformamide) was added
to each well and incubated for 2 h. The reduction of p-iodo-
nitrotetrazolium was spectroscopically measured at 450 nm
using TRIAD multimode reader (Dynex Technologies,
Chantilly, VA) to determine the minimum inhibitory con-
centration (MIC) values. All the results were represented as
the average of three independent experiments.
2.6. Growth analysis of S. aureus and E. coli
The growth rate profile of S. aureus and E. coli in the pre-
sence of Kocuran-capped AgNPs at different concentrations
was determined by measuring the optical density at 600 nm.
The optical density of 0.1 at 600 nm corresponded to a con-
centration of 108 cfu ml−1. The bacteria (OD600 of 0.1) were
cultured in Mueller-Hinton broth in the absence and presence
of Kocuran-capped AgNPs at different concentrations (0.1, 1,
5, and 10 μg ml−1); incubated at 37 °C for 24 h and the cell
viability (%) was assessed based on the colony forming units
(cfu ml−1) measurements. The results are represented as the
means of triplicate experiments.
2.7. TEM studies
The bacterial cultures were centrifuged at 5000 g and washed
thrice with 0.1 M HEPES buffer and then fixed overnight with
1% (v v−1) osmium tetroxide in HEPES buffer followed by
sequential gradient dehydration from 10% to 100% (v v−1)
ethyl alcohol and then TEM images were captured on FEI
Tecnai G2 S-Twin TEM (FEI Company, Hillsboro, OR)
operated at an accelerating voltage of 300 kV.
Nanotechnology 25 (2014) 325101
2.8. Measurement of hydroxyl radical formation in S. aureus
and E. coli
The induction of the formation of hydroxyl radicals in S.
aureus and E. coli cultures in the presence of Kocuran-capped
AgNPs was measured using the fluorescent reporter dye, 3′-
(p-hydroxyphenyl) fluorescein (HPF). The bacterial cultures
(OD600 of 0.1) were treated with Kocuran-capped AgNPs
(5 μg ml−1) and incubated at 37 °C for 3 h. Samples were
collected just before the addition of AgNPs and at periodic
intervals of 1 h, washed with distilled water and suspended in
PBS (pH 7.2). The hydroxyl radical formation was detected
using 5 mM of HPF by measuring the mean fluorescence on
FACS Caliber flow cytometer (BD Biosciences, Mountain
View, CA, USA).
2.9. Measurement of protein leakage in S. aureus and E. coli
The protein leakage from S. aureus and E. coli cells was
determined by treatment of the bacterial cultures (OD600 of
0.1) with Kocuran-capped AgNPs at a concentration of
5 μg ml−1 and incubated at 37 °C for 4 h. Sample aliquots
(1 ml) were collected periodically at hourly intervals and
centrifuged at 5000 g for 30 min at 4 °C. The protein content
in the cell-free supernatant was quantified using Bradford’s
assay [35]. The untreated cultures were used as controls and
all the experiments were run in triplicates.
2.10. Measurement of NAD+ and NADH concentration in S.
aureus and E. coli
The changes in NAD+ and NADH concentration in S. aureus
and E. coli cultures in the presence of Kocuran-capped
AgNPs was monitored by NAD cycling assay [36]. The S.
aureus and E. coli cultures were treated with silver glyco-
nanoparticles (5 μg ml−1) and incubated at 37 °C. Sample
aliquots (5 ml) were collected at periodic hourly intervals for
6 h, centrifuged at 10 000 g at 4 °C for 10 min and the cell
pellets were collected. To the ice-cold cell pellets, 75 μl of
0.2 M NaOH was added to extract NADH and 75 μl of 0.2 M
HCl was added for NAD+ extraction. The samples were then
heated at 100 °C for 10 min and centrifuged at 10 000 g for
5 min. The NAD+ cycling assay was performed in a 96-well
microtiter plate. The reaction mixture contained 30 μl of 1 M
Bicine buffer (pH 8.0), 75 μl sample, 75 μl neutralizing buffer
(0.1 M HCl for NADH, or 0.1 M NaOH for NAD+), 30 μl of
phenazine methosulfate, 30 μl of 3-(4, 5–dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT), 30 μl of 100%
ethanol and 30 μl of 40 mM EDTA. After incubation at 30 °C
for 3 min, 6 μl of yeast alcohol dehydrogenase I (ADH-I,
Sigma) at a concentration of 500 U ml−1 was added and the
rate of MTT reduction was recorded by measuring the
absorbance at 570 nm. The rate of MTT reduction is pro-
portional to the concentration of NAD+ or NADH in the
sample.
4
C G Kumar and P Sujitha
2.11. Biofilm assay in S. aureus and E. coli
The effect of Kocuran-capped AgNPs on the biofilm formation
of S. aureus and E. coli cultures was assessed by crystal violet
staining [37]. The cultures were initially incubated in 96-well
microtiter plates at 37 °C for 12 h. The cultures were treated
with Kocuran-capped AgNPs at different concentrations (0.1,
1, 5 and 10 μg ml−1) and re-incubated at 37 °C for 24 h. The
biofilm formation was assessed by staining with 0.1% crystal
violet for 5 min and the dye was solubilized with 150 μl of
isopropanol-0.04 N HCl and 50 μl of 0.25% SDS and the plates
were spectroscopically measured at 590 nm. The percentage of
biofilm formation was calculated from the equation: [1 − (ODT/
ODC)] × 100 where ODT is the optical density of the cultures
treated with Kocuran-capped AgNPs and ODC is the optical
density of untreated cultures. All the results were represented
as the average of three independent experiments.
2.12. Confocal laser scanning microscopy
The biofilm formation was assessed using LIVE/DEAD®
BacLight™ Bacterial Viability kit and visualized by confocal
laser scanning microscopy. Briefly, the cells of S. aureus and
E. coli (OD600 = 0.01) were cultured on Thermanox plastic
cover slips placed in the wells of 24-well plates containing
Muller–Hinton broth for 12 h at 37 °C. Later, they were
treated with Kocuran-capped AgNPs at different concentra-
tions (0.1, 1, 5 and 10 μg ml−1) for 24 h. The cover slips were
washed with PBS and the bacterial biofilms were stained with
1 ml of 0.6% Live/Dead BacLight stain following the staining
kit instructions. The stained biofilms were visualized by
Olympus FluoView 500 confocal laser microscope (Olympus
Optical, Tokyo, Japan) using an argon ion laser at
480–490 nm for excitation and a 500–635 nm band pass filter
for emission. CLSM images were processed by Olympus
FluoView 500 software and the scale bar for each image
is 10 μm.
2.13. Biofilm inhibition assay on silicone urethral catheters
Silicone urethral catheters were coated with the Kocuran-
capped AgNPs according to a simple dispersion method. A
sterile silicone urethral catheter (Unomedical A/S, Birkerod,
Denmark) was cut into 4 cm long pieces and these pieces
were placed in test tubes incubated in the colloidal solution of
Kocuran-capped AgNPs (100 μg) for 24 h at 37 °C. The
catheters were later completely dried at 50 °C to form an
antimicrobial coating. The time-dependent performance of the
antimicrobial coating of Kocuran-capped silver glyconano-
particles on silicone urinary catheters was evaluated against
uropathogenic strains of S. aureus and E. coli which were
grown overnight in test tubes with sterile silicone urethral
catheters coated with and without Kocuran-capped AgNPs
(100 μg) and the tubes were then incubated for 24, 48, 72 and
96 h at 37 °C. The cultures were removed and the catheters
were washed with distilled water. After washing, 3 ml of
crystal violet (0.1%) was added to the catheters and stained
for 10 min. The stained biofilms were rinsed with distilled
water (3×) and allowed to dry at room temperature for 15 min
Nanotechnology 25 (2014) 325101
before examination. The dye was solubilized with 150 μl of
isopropanol-0.04 N HCl and 50 μl of 0.25% SDS and the
collected dye solution was read at 590 nm on a UV–visible
double beam spectrophotometer (Lambda 25, PerkinElmer,
Shelton, CT, USA). The results were expressed in percent
inhibition of growth of free-floating organisms or biofilm
formation in each test tube relative to the average growth or
biofilm formation in control tubes (tubes with untreated
catheters). The results were represented as the means of tri-
plicate experiments. High resolution-scanning electron
microscope (HR-SEM) images were also recorded using a
Hitachi S-4700 Scanning Electron Microscope (Hitachi High
Technologies, Pleasanton, CA) to determine the surface
topography of biofilm formation on untreated and treated
catheters coated with Kocuran-capped AgNPs.
2.14. In vitro biocompatibility studies of Kocuran-capped silver
glyconanoparticles
The in vitro biocompatibility of Kocuran-capped silver gly-
conanoparticles was evaluated on human gingival fibroblast
(HGF) cell line procured from ScienCell Research Laboratories
(Carlsbad, CA, USA) using 3-(4,5-dimethylthiazol-2yl)-2,5-
diphenyl tetrazolium bromide (MTT) reduction method [38].
The HGF cell line was grown in DMEM supplemented with
10% fetal bovine serum and 100 IU ml−1 penicillin-strepto-
mycin and incubated at 37 °C in 5% CO2 humidified atmo-
sphere. For assessing the in vitro biocompatibility, the HGF
were cultured at a cell density of 5 × 103 cells well−1 in 96 well
plates for 12 h and then treated with Kocuran, Kocuran-capped
silver glyconanoparticles and commercially available citrate-
synthesized AgNP at various concentrations (1–200 μg ml−1).
The cells were incubated at 37 °C for 48 h and then 20 μl of
MTT solution (5 mg ml−1 in PBS) was added to each well and
again incubated for 4 h at 37 °C. The purple colored formazan
crystals formed were dissolved in 150 μl well−1 of DMSO and
the cell proliferation (%) was measured at 570 nm in a
microplate reader (BioRad Laboratories, Hercules, CA, USA).
The results are represented as the means of triplicate experi-
ments run in four replicates.
2.15. Statistical analysis
Statistical analysis was performed using GraphPad PRISM
software version 3.0 (GraphPad Software, La Jolla, CA,
USA). Data are expressed as the mean ± S.D of three inde-
pendent experiments. All experimental data were compared
using Student’s t-test. In all comparisons, p < 0.05 was con-
sidered statistically significant.
3. Results and discussion
3.1. Synthesis and characterization of Kocuran-capped silver
glyconanoparticles
Preliminary optimization experiments were carried out to
determine the optimal concentrations of Kocuran (supple-
mentary figure S1) and silver nitrate (supplementary figure S2)
5
C G Kumar and P Sujitha
that would have an influence on the size, shape or stability.
Based on the optimization studies, the optimal concentrations
to be used were Kocuran (10 mg ml−1) and AgNO3 (100 mM).
The bioreduction of silver ions was monitored by UV-visible
spectroscopy and the spectra for the reaction of cell-free
supernatant with silver nitrate were recorded at room tem-
perature (28 °C) as a function of time (supplementary figure
S3). The reaction due to the reduction of silver ions was evi-
denced by the development of yellowish brown color. This
could be attributed to the direct redox reaction between
Kocuran and silver nitrate, since there was no addition of any
reducing agent in the reaction mixture. The nanoparticles
exhibited an absorption peak around 435 nm after 6 min of
reaction, which is a characteristic SPR band of silver nano-
particles possibly due to the excitation of longitudinal plasmon
vibrations in silver nanoparticles in the solution [39, 40]. It was
also observed that the absorption peak intensity increased as a
function of time and varied with increased Kocuran con-
centration (data not shown). This variation can be attributed to
the difference in particle size and shape of the synthesized
nanoparticles [41, 42]. Further, the increased intensity and
sharpness in the SPR band of excitation spectra indicated that
the silver nanoparticles were effectively capped and/or stabi-
lized with Kocuran and were non-agglomerated. In the case of
polysaccharide-based nanoparticle synthesis, it was demon-
strated that the sugar moieties of the EPS molecules mainly
contribute to the reducing effect and can also act as effective
colloidal stabilizers that can be explored for green synthesis of
nanoparticles [43].
The proposed mechanism of reduction in the case of
Kocuran can be explained as follows: Kocuran is an acidic
tetrapolysaccharide with repeating sugar residues of glucose,
galactose, mannose (sugar content 91%) and glucuronic acid
content (9%) [32]. The hydroxyl and carboxylic groups of the
sugar moieties present in this biopolymer assists in the
complexation of silver ions. The silver ions oxidize the
hydroxyl groups to carbonyl groups and during this process
the silver ions are reduced to elemental silver. In addition to
this inherent oxidation, the dissolved air may also cause
oxidation of the existing hydroxyl groups to carbonyl groups
such as aldehydes and carboxylates. These potential reducing
aldehyde groups along with the other existing carbonyl
groups reduce more and more of silver ions to elemental
silver. In this process, the nanoparticles are probably capped
and stabilized by the EPS molecules. Since these carbohy-
drate polymers are very complex, it is expected that more than
one mechanism is involved in the complexation and sub-
sequent reduction of silver ions by the Kocuran poly-
saccharide. Complexation of silver ions by hydroxyl groups
and its subsequent reduction by aldehyde groups was earlier
reported in the case of starch-capped silver nanoparticles [44].
In the case of silver nanoparticles produced using gum Aca-
cia, the carboxylate groups were involved in the complexa-
tion of silver ions and its subsequent reduction by hydroxyl
groups [45].
The FT-IR spectra revealed that the surface functional
groups present on the Kocuran-capped AgNPs and that of
pure Kocuran. The absorption peak position observed at
Nanotechnology 25 (2014) 325101
Figure 1. Transmission electron micrograph of Kocuran-capped
silver glyconanoparticles dispersed on an amorphous carbon-coated
copper grid. Scale bar corresponds to 20 nm. Inset: selected electron
area diffraction (SAED) pattern of silver glyconanoparticles.
3429 cm−1 corresponded to the hydroxyl stretch, while the
absorption peaks observed at 2924 cm−1 represented the C–H
stretching. The peaks at 1073 and 693 cm−1 were attributed to
C–O–C and C–O-P rings of polysaccharides (supplementary
figure S4(a)). After the bioreduction of silver nitrate with EPS
sugars in the solution phase, the created peak at 1636 cm−1 is
characteristic to the binding of the –C = O functional moiety
and the shift in the peak at 1032 towards a lower frequency as
compared to the peak positioned at 1073 cm−1 for Kocuran is
attributed to the binding of C-O-C and C-C-H groups of the
Kocuran EPS with silver nanoparticles. Further, the broad
peak at 635 cm−1 is related to the silver nanoparticles bonding
with oxygen from the hydroxyl groups of the sugar residues
(supplementary figure S4(b)). Based on these observations,
the FT-IR spectra clearly suggests that Kocuran molecules
capped on the silver nanoparticles and the hydroxyl groups
played a key role in reducing and stabilizing the silver
nanoparticles. Our results corroborates with the observations
of the earlier reports [46, 47].
To gain a detailed understanding of the size and shape of
silver glyconanoparticles, the surface morphology of the
Kocuran-capped AgNPs was examined by TEM. The TEM
image (figure 1) clearly revealed that the Kocuran-capped
AgNPs were found to be non-agglomerated, predominantly
were spherical in shape and mono-dispersed. The average
particle size and size distribution (polydispersity index) of the
AgNPs were determined by DLS analysis and the average
size of nanoparticles computed from the histogram was found
to be 12 nm with a narrow size distribution (supplementary
figure S5). The electrostatic repulsion between the nano-
particles depends on the zeta potential value (measured in
mV). It is observed that the higher the zeta potential, the
stronger the repulsion which results in a more stable system
[48]. Therefore, the zeta potential value aids in understanding
6
C G Kumar and P Sujitha
Figure 2. XRD spectrum of Kocuran-capped silver
glyconanoparticles.
the state of the nanoparticle surface and predicting its long-
term stability. Kocuran-capped AgNPs were stable and
showed no agglomeration which was supported by the zeta
potential value of −33.9 mV and were anionic charged (sup-
plementary figure S6). It was reported that nanoparticles with
zeta potential values greater than +30 mV or lower than
−30 mV typically have high degrees of stability. However, the
dispersions with a low zeta potential will eventually aggregate
due to van der Waals interparticle attraction [48]. The EDAX
spectral analysis revealed the elemental composition profile of
the synthesized Kocuran-capped AgNPs, indicating the pre-
sence of strong elemental silver signals at 3 keV (supple-
mentary figure S7). Furthermore, the XRD spectrum of the
Kocuran-capped AgNPs (figure 2) showed four main char-
acteristic Bragg diffraction peaks for silver nanocrystallites at
2θ values of 40.1, 45.1, 65.0, and 82.1 which corresponded to
the main (111), (200), (220) and (311) crystallographic planes
of the face-centered cubic (fcc) silver crystals, which con-
firmed the SAED results. The size, shape, stability and
bioactivity are mainly dependent on the type of the capping
ligand used for nanoparticle synthesis. Different natural
polysaccharides such as starch [44], gum acacia [45], chitosan
[49], and gum ghatti (Anogeissus latifolia) [50] have been
earlier explored for the synthesis of silver nanoparticles.
Recent reports suggests that diverse polysaccharides of
microbial origin such as carboxymethylated curdlan and
fucoidan biopolymers [51], sulphated polysaccharides iso-
lated from marine red algae, Porphyra vietnamensis [52],
exopolysaccharides from Lactobacillus rhamnosus [53] and
B. subtilis [47] have gained renewed interest as promising
capping ligands for the biosynthesis of AgNPs for different
therapeutic applications.
3.2. Stability analysis of Kocuran-capped silver
glyconanoparticles
It was earlier reported that the instability of silver nano-
particles at different physical and chemical conditions leads to
Nanotechnology 25 (2014) 325101 C G Kumar and P Sujitha

the dissociation of ions, formation of aggregates and increase Table 1. Antimicrobial activities of Kocuran, Kocuran-capped silver
in the particle size that affects the electromagnetic properties, glyconanoparticles and commercially available silver nanoparticles.
mobility and bioavailability of silver nanoparticles [54]. Minimum inhibitory concentration (MIC,
Considering these facts, the effect of pH, temperature, solu- μg ml−1)
tions of NaCl, NaNO3 and BSA on the stability of Kocuran-
Kocuran-capped
capped AgNPs was studied by measuring the change in the Bacterial silver glyconano- Silver nano-
SPR absorption band using UV-visible spectroscopy. The strainsa Kocuran particles particlesb
Kocuran-capped AgNPs were stable at a pH range of 7 to 10
(supplementary figure S8(a)) and temperature range of 30 °C Staphylococcus 150 4.68 9.37
to 100 °C (supplementary figure S8(b)). In both the cases, aureus
there was no significant change observed in their absorption MTCC 96
S. aureus 150 9.37 18.75
spectrum. In addition, the Kocuran-capped AgNPs exhibited
MLS16
good stability when dispersed in electrolyte solutions of NaCl MTCC 2940
and NaNO3 at concentrations of 10, 50, 100 and 150 mM Bacillus subtilis 75 18.75 18.75
(supplementary figures S8(c), (d)). However, at 200 mM MTCC 121
concentration of NaCl and NaNO3, there was a slight decrease Micrococcus 75 37.5 75
in the absorption intensity. The AgNPs also showed similar luteus
absorption spectrum in BSA solutions of 0.05, 0.1, 0.2 and MTCC 2470
0.3%, while a slight decrease in the absorption intensity was Klebsiella plan- 150 18.74 18.74
observed in the case of 0.4 and 0.5% BSA solutions (sup- ticola
plementary figure S8(e)). The slight decrease in the absorp- MTCC 530
tion intensity at higher concentration (>200 mM) for Escherichia coli 75 4.68 9.37
MTCC 739
electrolyte solutions like NaCl and NaNO3, and BSA solution
Pseudomonas 150 37.5 75
concentrations (0.4% and 0.5%) is mainly due to the aggre- aeruginosa
gation of nanoparticles [55]. These stability studies clearly MTCC 2453
demonstrate that the Kocuran-capped AgNPs under different E. coli ATCC 300 37.5 75
conditions of pH, temperature, NaCl, NaNO3 and BSA 35218
solutions qualifies it as a promising candidate for various S. aureus subsp. 300 18.74 75
biological and therapeutic applications. The stability of the aureus ATCC
Kocuran-capped AgNPs was evaluated by UV-visible spec- 29213
troscopy and the respective absorption spectra were recorded Enterococcus 300 18.74 18.74
after 1 h, 5 days, 10 days, 15 days, 30 days, 45 days and 60 faecalis
days of storage at room temperature (supplementary figure S8 ATCC 29212
(f)). The UV-visible spectra showed that the Kocuran capped a
MTCC = Microbial type culture collection, CSIR-Institute of Microbial
AgNPs exhibited an intense SPR peak around 450 nm. As the Technology, Chandigarh, India; ATCC = American type culture collection,
time progressed from 1 to 30 days, similar plasmon resonance Manassas, VA, USA.
b
bands were observed with no change in the position of SPR, Commercial AgNP (citrate-reduced) procured from NanoComposix, San
Diego, CA, USA.
except for a decrease in the absorption intensity. After 30th
day, a shift in the position of SPR peak was observed with a
decrease in intensity, suggesting that the stability of nano-
particles decreased after 30 days. 18.74 and 18.74 μg ml−1, while the commercial AgNP
showed MIC values of 75, 75 and 18.74 μg ml−1. In com-
parison to commercial AgNP, the Kocuran-capped silver
3.3. Antimicrobial activity studies of Kocuran-capped silver
glyconanoparticles exhibited good antimicrobial activity
glyconanoparticles
which qualifies them as a promising antimicrobial agent
Kocuran-capped AgNPs exhibited good antibacterial activity against multidrug resistant bacterial pathogens.
against both Gram-positive and Gram-negative bacterial The MIC value of a particular antimicrobial agent is an
strains. The minimum inhibitory concentrations observed important quantitative parameter dependent on the microbial
against different bacterial pathogens and drug resistant strains strain. Different factors such as particle size, shape, crystal-
are shown in table 1. Kocuran-capped AgNPs exhibited linity, surface chemistry, pH, ionic strength, divalent cations,
promising antimicrobial activity against S. aureus MTCC 96 ligands and macromolecules were demonstrated to affect the
and E. coli MTCC 739 with MIC values of 4.68 μg ml−1, and antimicrobial activity [56]. In the present study, Kocuran and
S. aureus MLS16 MTCC 2940 (MIC value of 9.37 μg ml−1), the corresponding Kocuran-capped silver glyconanoparticles
while moderate antimicrobial activity was observed against exhibited different MIC values with regard to different bac-
M. luteus MTCC 2470 and P. aeruginosa MTCC 2453 with terial strains. This may be due to the fact that when Kocuran
MIC values of 37.5 μg ml−1. The MIC values for the drug and the corresponding Kocuran-capped silver glyconano-
resistant strains such as E. coli ATCC 35218, S. aureus subsp. particles interacted with the test strains; the bacterial strains
aureus ATCC 29213 and E. faecalis ATCC 29212 were 37.5, counteract the effect by secreting secondary metabolites into

7
Nanotechnology 25 (2014) 325101
the medium which alter the pH and ionic strength. These
variations influence the interaction and binding of nano-
particles to the bacterial cell and also the aggregation states of
silver nanoparticles. The microbial strains under these con-
ditions exhibited differences in the vulnerability to silver
nanoparticles and hence the differences in MIC values were
observed against the tested bacterial strains.
Further, the antimicrobial activity effect exhibited by
Kocuran-capped silver nanoparticles is additive since the
silver nanoparticles synthesis is without the addition of any
external reducing agent and Kocuran itself acted as a capping
ligand causing the reduction and stabilization of the silver
nanoparticles. In order to prove that Kocuran-capped glyco-
nanoparticles contributed to the antimicrobial activity,
experiments were performed in comparison with commer-
cially available citrate-synthesized silver nanoparticles. The
results suggest that Kocuran-capped glyconanoparticles
showed good antimicrobial activity as compared to citrate-
synthesized silver nanoparticles. It was earlier reported that
EPS capped AgNPs exhibited efficient bactericidal activity
and higher adherence towards the bacterial cell surface. The
physical interaction between the nanoparticles and the bac-
terial cell wall surface was found to be the key factor for cell
damage [47]. Similarly, in the present study, the Kocuran
polysaccharide capped on the AgNPs might have contributed
to the better interaction with the bacterial cell wall and also
enhanced the effective anchoring of AgNPs on the cell sur-
face. The AgNP anchored to the bacterial cell surface serve as
reservoirs for silver ions and the release of these silver ions
causes cell death.
In the present study, S. aureus MTCC 96 and E. coli
MTCC 739 strains exhibited promising antimicrobial activity
and they were selected for further studies as representative
Gram-positive and Gram-negative bacterial strains, which are
the most common pathogenic bacteria. It was earlier reported
that silver and nanosilver exhibited effective antimicrobial
activity against different pathogenic species such as E. coli, B.
subtilis, Vibrio cholerae, S. aureus, P. aeruginosa and
Streptococcus pyrogens [57]. The mechanism of antibacterial
effect of nanosilver is complex and the cell death is majorly
caused by direct damage of the cell membrane, uncoupling
oxidative phosphorylation, induced free radical formation and
interfering with the respiratory chain, electron chain system,
proteins, membrane-bound enzymes and DNA [56].
3.4. Effect of Kocuran-capped silver glyconanoparticles on the
growth of S. aureus and E. coli
The effect of Kocuran-capped AgNPs at concentrations of
0.1, 1, 5 and 10 μg ml−1 on the growth of S. aureus MTCC 96
and E. coli MTCC 739 was assessed as a function of time and
the growth curves are represented as supplementary figures
S9(a), (b). The untreated control group of S. aureus and
E. coli exhibited normal growth pattern as observed by a
steady increase in the optical density at 600 nm over the
incubation time. When treated with 0.1 μg ml−1 of AgNPs
there was no significant change in the growth for both S.
aureus and E. coli cultures, while treatments with 5 and
8
C G Kumar and P Sujitha
10 μg ml−1 showed a significant decrease in the cell viability
(cfu ml−1) and the growth was completely inhibited after 2 h
of post-treatment period, in the case of both S. aureus and
E. coli cultures. This observation was further confirmed by
TEM analysis (figure 3), which revealed that in the case of
both S. aureus and E. coli cultures treated with Kocuran-
capped AgNPs (5 μg ml−1), the intracellular localization of
AgNPs was observed which exerted the possible bactericidal
action.
3.5. Effect of Kocuran-capped silver glyconanoparticles on
hydroxyl radical production in S. aureus and E. coli
The ability of Kocuran-capped AgNPs to induce reactive
oxygen species (ROS) production was monitored by mea-
suring the production of hydroxyl radicals. The hydroxyl
radical production in S. aureus MTCC 96 and E. coli MTCC
739 in the presence of silver glyconanoparticles at a lethal
concentration (5 μg ml−1) was measured as a function of time
using 3′-(p-hydroxyphenyl) fluorescein (HPF) fluorescence.
In the case of untreated cultures of S. aureus and E. coli, there
was no significant increase in the hydroxyl radical production
over the post-treatment period as indicated by a constant HPF
fluorescence (figures 4(a), (c)). When S. aureus and E. coli
cultures were treated with Kocuran-capped AgNPs, there was
an increase in the HPF fluorescence during the post-treatment
period indicating the generation of hydroxyl radicals
(figures 4(b), (d)). This steady increase in the hydroxyl radi-
cals in both S. aureus and E. coli is lethal to the bacterial cells
which results in a decreased absorbance (confirmed by growth
curve analysis). This suggests the probable adverse role of
ROS in the primary generation of hydroxyl radicals by
Kocuran-capped AgNPs which results in lethal and lytic
effects in the case of both S. aureus and E. coli cells.
3.6. Effect of Kocuran-capped silver glyconanoparticles on
microbial electron transport system
The effect of Kocuran-capped AgNPs on the microbial elec-
tron transport chain was assessed by measuring the change in
the levels of NAD+/NADH ratios through NAD cycling
assay. The change in the ratios of NAD+ and NADH (NAD+/
NADH) in S. aureus MTCC 96 and E. coli MTCC 739 during
the post-treatment of Kocuran-capped AgNPs is shown in
figures 5(a), (b). After 1 h of post-treatment with AgNPs
(5 μg ml−1), a predominant increase in NAD+/NADH ratios to
2.26 and 2.83 was observed in the case of both S. aureus and
E. coli, respectively, while in the case of untreated cultures,
the NAD+/NADH ratios remained at a constant level of 0.47
and 0.32 for both S. aureus and E. coli cultures, respectively.
Kocuran-capped AgNPs affected the microbial electron
transport system as indicated by the increase in NAD+/NADH
ratios which is due to increased oxidation of NADH through
the electron transport chain. Further, the hyperactivation of
electron transport chain and rapid depletion of reducing
equivalents resulted in superoxide production [58, 59]. It was
established that the superoxide causes damage to the iron-
sulphur clusters of the redox system making it susceptible to
Nanotechnology 25 (2014) 325101 C G Kumar and P Sujitha

Figure 3. TEM analysis of (a) S. aureus MTCC 96 and (b) E. coli MTCC 739 treated with Kocuran-capped silver glyconanoparticles. The
organisms were cultured in Muller–Hinton broth and treated with Kocuran-capped silver glyconanoparticles (5 μg ml−1) and further
incubated at 37 °C for 24 h. The intracellular localization of Kocuran-capped silver glyconanoparticles was observed through TEM images
(A1 and A2 of S. aureus MTCC 96, and B1 and B2 of E. coli MTCC 739). Scale bar of A1 and B1 is 1 μm, while A2 and B2 are magnified
images of A1 and B1 at scale bar of 200 nm.

Fenton reaction. These ferrous ions are oxidized through the
Fenton reaction forming hydroxyl radicals [60].
3.7. Effect of Kocuran-capped silver glyconanoparticles on
protein leakage in S. aureus and E. coli
The bactericidal action of Kocuran-capped AgNPs causing
protein leakage was monitored by measuring the release of
protein content from the bacterial cells during the post-treat-
ment period. The extent of protein leakage in S. aureus
MTCC 96 and E. coli MTCC 739 cells when treated with
Kocuran-capped AgNPs at 5 μg ml−1 is depicted in supple-
mentary figures S10(a), (b). After 1 h of post-treatment, there
was no significant increase in the protein leakage in the case
of both S. aureus and E. coli cells as compared to untreated
cultures. After 2 h of post-treatment, the protein leakage
increased from 27.9 μg ml−1 to 45.7 μg ml−1 in the case of S.
aureus, while the protein leakage in the case of E. coli
increased from 21.1 μg ml−1 to 50.18 μg ml−1. However, the
untreated S. aureus and E. coli cultures showed no increase in
protein leakage. This increased protein leakage from the
bacterial cultures upon treatment to lethal doses of Kocuran-
capped silver glyconanoparticles correlates to the decline in
the viability of S. aureus and E. coli cells. It was earlier
reported that hydroxyl radicals are extremely toxic and exert
deleterious effect on proteins, DNA, lipids and other bio-
molecules. They react with the membrane lipids to cause lipid
peroxidation and increased membrane permeability that
results in protein leakage and subsequent cellular death [61].
3.8. Antibiofilm activity of Kocuran-capped silver
glyconanoparticles
Kocuran-capped AgNPs effectively inhibited the biofilm
formation by S. aureus MTCC 96 and E. coli MTCC 739 in a
concentration-dependent manner (figure 6). Kocuran-capped
AgNPs at 1 μg ml−1 concentration reduced the preformed
biofilm formation by S. aureus and E. coli to 61.44% and
58.16%, respectively. When the biofilms were treated with
5 μg ml−1 of AgNPs, the biofilm formation was 20.08% and
18.17% as compared to the untreated cultures. In order to
check the antibiofilm activity, the biofilms were stained with
LIVE/DEAD BacLight bacterial viability kit and the confocal
laser scanning microscope (CLSM) images showed a dose-
dependent inhibitory effect of Kocuran-capped AgNPs on the
biofilm formation by S. aureus MTCC 96 (figure 7) and

9
Nanotechnology 25 (2014) 325101 C G Kumar and P Sujitha

Figure 4. Effect of Kocuran-capped silver glyconanoparticles on hydroxyl radical production in S. aureus MTCC 96 and E. coli MTCC 739.
The hydroxyl radical production was measured using the HPF fluorescence as a function of time in (a) S. aureus MTCC 96 without Kocuran-
capped silver glyconanoparticles treatment (control). (b) S. aureus MTCC 96 treated with Kocuran-capped silver glyconanoparticles. (c)
E. coli MTCC 739 without Kocuran-capped silver glyconanoparticles treatment (control). (d) E. coli MTCC 739 treated with Kocuran-
capped silver glyconanoparticles. The S. aureus MTCC 96 and E. coli MTCC 739 bacterial cells were cultured in Muller–Hinton broth in the
presence of Kocuran-capped silver glyconanoparticles (5 μg ml−1) and incubated at 37 °C for 24 h and the mean HPF fluorescence was
measured. The S. aureus MTCC 96 and E. coli MTCC 739 cultures without treatment of Kocuran-capped silver glyconanoparticles were run
in parallel as controls. The peaks represent hydroxyl radical formation at 1 h (black line), 2 h (blue line), 3 h (green line) and 4 h (red line)
after post-treatment, respectively.

E. coli MTCC 739 (figure 8). The LIVE/DEAD BacLight kit
is used to monitor the viability of bacterial populations as a
function of the membrane integrity of the cells which uses
appropriate mixtures of SYTO 9, a green fluorescent nucleic
acid dye, and propidium iodide, a red fluorescent nucleic acid
dye. The SYTO 9 is permeable to live bacterial cells having
intact cell membranes and stains them green, while the pro-
pidium iodide stains red the bacteria having damaged cell
membranes that are considered to be dead or in dying state.
Under CSLM, the images are thus observed as green and red
fluorescent images for live and dead bacteria, respectively.
The untreated S. aureus and E. coli cells showed rapid biofilm
formation. There was no significant change in the biofilm
formation by S. aureus and E. coli when they were treated
with 0.1 μg ml−1 of Kocuran-capped AgNPs. Further, when
treated with 1 μg ml−1 of AgNPs, the intact architecture in S.
aureus and E. coli was changed. The treatment with 5 and
10 μg ml−1 of AgNPs damaged the structural integrity of the
biofilm and caused bacterial cell death. These results reflect
the strong inhibitory effect of Kocuran-capped AgNPs on
biofilm formation by S. aureus and E. coli.
3.9. Antibiofilm property of Kocuran-capped silver
glyconanoparticle coating on silicone urethral catheters
The proposed mechanism of Kocuran-capped silver glyco-
nanoparticle-coating on the silicone urethral catheters can be
explained as follows: surface charge and hydrophobicity are
the two properties that play a critical role in determining the
surface chemistry of the coating material that will influence
the bacterial attachment and biofilm formation [62]. The
Kocuran-capped silver glyconanoparticles are negatively
charged based on the zeta potential value and the coatings on
the hydrophobic silicone urethral catheters are due to elec-
trostatic interactions which in turn repel bacterial adhesion
due to electrostatic repulsion between the often negatively

10
Nanotechnology 25 (2014) 325101
Figure 5. Effect of Kocuran-capped silver glyconanoparticles on
NAD+/NADH ratio in S. aureus MTCC 96 and E. coli MTCC 739.
The NAD+/NADH ratios were measured as a function of time in (a)
S. aureus MTCC 96 and (b) E. coli MTCC 739 in the presence of
Kocuran-capped silver glyconanoparticles. The S. aureus MTCC 96
and E. coli MTCC 739 were cultured in Muller–Hinton broth in the
presence of Kocuran-capped silver glyconanoparticles (5 μg ml−1)
and incubated at 37 °C for 24 h and the levels of NAD+/NADH
ratios were measured by NAD cycling assay. The untreated group
represents the S. aureus MTCC 96 and E. coli MTCC 739 cultures
that did not receive treatment with Kocuran-capped silver glycona-
noparticles. All the experiments were repeated three times and the
bars represent the means ± S.D.
Figure 6. Biofilm formation by S. aureus MTCC 96 and E. coli
MTCC 739 treated with Kocuran-capped silver glyconanoparticles.
The S. aureus MTCC 96 and E. coli MTCC 739 were cultured in
Muller–Hinton broth for 12 h at 37 °C and then treated with 0.1, 1, 5
and 10 μg ml−1 of Kocuran-capped silver glyconanoparticles for
24 h. The biofilm formation was demonstrated using crystal violet
assay. The untreated group represents the S. aureus MTCC 96 and
E. coli MTCC 739 cultures that did not receive treatment with
Kocuran-capped silver glyconanoparticles. All the experiments were
repeated three times and the bars represent the means ± S.D.
11
C G Kumar and P Sujitha
charged bacterial surface and the negatively charged glyco-
nanoparticle coated catheter surface. Further, the time-
dependent performance evaluation of Kocuran-capped silver
glyconanoparticle-based coating on silicone urethral catheters
showed inhibition in the biofilm formation by S. aureus and
E. coli after 24, 48, 72 and 96 h incubation as revealed by the
crystal violet assay and illustrated in figures 9 and 10. The
antimicrobial effect on biofilm formation by Kocuran-capped
silver glyconanoparticle-based coating on silicone urethral
catheters as a function of time showed that the biofilm for-
mation in Kocuran-capped AgNPs-coated catheters was 9.1%
and 10.7%, while in the case of uncoated catheters was
89.99% and 93.24%, respectively, after 24 h incubation, as
quantified by crystal violet assay. Moreover, the Kocuran-
capped silver glyconanoparticles-coated catheters showed
reduced biofilm formation of 13.78% and 14.29% in S. aur-
eus and E. coli, respectively, even after 96 h of incubation,
while the uncoated catheters showed 99% biofilm formation
in the case of both S. aureus and E. coli (supplementary
figures S11(a), (b)). Thus, the catheters functionalized with
Kocuran-capped silver glyconanoparticles acted as anti-
microbial coatings which were effective in inhibiting the
bacterial adhesion and preventing the biofilm formation by
uropathogenic bacterial strains like S. aureus MTCC 96 and
E. coli MTCC 739.
The HR-SEM analysis to reveal the surface topography
confirmed that the AgNPs-coated catheters showed inhibition
of biofilm formation by S. aureus and E. coli (figure 11),
while the biofilm formation was profuse in the case of
uncoated catheters (figure 12). Intensive care units are the
common sites for transmission of nosocomial bacterial
infections which result in serious complications after surgery
[63]. The inner and outer abiotic surfaces of several biome-
dical devices such as stents, heart valves, vascular grafts and
catheters are frequently colonized with bacteria through
bacterial adhesion and biofilm formation resulting in these
infections [64], which is a widely growing problem in health
care system. It was earlier demonstrated that the coating of
AgNPs on polyurethane and plastic catheters exhibited anti-
bacterial and antiadhesive properties against pathogenic
strains of E. coli, Enterococci, P. aeruginosa and Staphylo-
coccus [65]. In the present study, the Kocuran-capped AgNPs
offered homogenous coating on the urethral catheters, which
effectively prevented the bacterial adhesion and biofilm for-
mation by S. aureus and E. coli. The results obtained in our
study clearly demonstrate the potential use of Kocuran-cap-
ped AgNPs on the surface coatings of the medical devices for
preventing bacterial infections.
3.10. In vitro biocompatibility studies of Kocuran-capped silver
glyconanoparticles
In the biodirected synthetic approach, the polysaccharide-
mediated synthesis and subsequent surface chemistry changes
of silver nanoparticles would increase the biocompatibility of
silver nanoparticles. The results of MTT assay are shown in
figure 13. The HGF cells incubated with Kocuran-capped
silver glyconanoparticles maintained higher average cell
Nanotechnology 25 (2014) 325101 C G Kumar and P Sujitha

Figure 7. Confocal laser scanning microscope (CLSM) analysis of biofilm formation by S. aureus MTCC 96 cells treated with Kocuran-
capped silver glyconanoparticles. The CLSM images of biofilm formation in S. aureus MTCC 96 (a) without treatment (control); (b) treated
with Kocuran-capped silver glyconanoparticles, 0.1 μg ml−1; (c) treated with Kocuran-capped silver glyconanoparticles, 1 μg ml−1; (d) treated
with Kocuran-capped silver glyconanoparticles, 5 μg ml−1; and (e) treated with Kocuran-capped silver glyconanoparticles, 10 μg ml−1. The S.
aureus MTCC 96 cells were cultured in Muller–Hinton broth on Thermanox plastic cover slips at 37 °C for 24 h and then treated with 0.1, 1,
5 and 10 μg ml−1 of Kocuran-capped silver glyconanoparticles. The biofilm formation was observed by CLSM after staining with Live/Dead
staining kit. The untreated S. aureus MTCC 96 represents the group that did not receive any treatment. The scale bars are 10 μm.

viabilities as compared to the commercial AgNP up to a
concentration of 100 μg ml−1. However, both types of Ag
nanoparticles significantly reduced the cell viability after 24 h
of incubation at a concentration range of 100–200 μg ml−1. In
addition, the biocompatibility of the polysaccharide
(Kocuran) alone tested at concentrations ranging from
1–200 μg ml−1 showed no significant difference in the cell
proliferation as compared to the untreated cells. In light of the
above, the MTT cell proliferation results demonstrated good
in vitro biocompatibility of the Kocuran-capped silver gly-
conanoparticles in human gingival fibroblasts as compared to
the commercial AgNPs suggesting that they are biocompa-
tible and non-toxic in nature and find suitable application in
urethral catheters.
4. Conclusions
We have developed a simple, rapid, clean and green chem-
istry approach for the synthesis of biocompatible silver gly-
conanoparticles using Kocuran EPS which acts as both a
capping and reducing agent. The biosynthesized AgNPs have
been thoroughly characterized using several spectroscopic
techniques. They were also assessed for their morphology,
stability, antibacterial and antibiofilm activities, induction of
intracellular ROS, depletion of NADH equivalents, and pro-
tein leakage. Kocuran-capped AgNPs were spherical with
narrow size distribution and were found to be stable and
showed no aggregation under various conditions of pH,
temperature, and in solutions of NaCl, NaNO3 and BSA. The
synthesized Kocuran-capped AgNPs exhibited pronounced
in vitro antibacterial and antiadhesive activities against the S.
aureus and E. coli strains in a concentration-dependent
manner. The bactericidal activity studies revealed that the
formation of bactericidal ROS, in particular hydroxyl radicals
in both S. aureus and E. coli, which contributed to the bac-
tericidal action of Kocuran-capped AgNPs. These hydroxyl
radicals subsequently activated the downstream signaling
pathways resulting in the depletion of reducing equivalents
and bacterial cell death. The enhanced antibacterial activity of
Kocuran-capped AgNPs may be attributed to the surface
functionalization with the EPS sugar residues, which facil-
itates the distribution and penetration of glyconanoparticles
into the cell wall, and the structure-activity relationship. In

12
Nanotechnology 25 (2014) 325101 C G Kumar and P Sujitha

Figure 8. Confocal laser scanning microscope (CLSM) analysis of biofilm formation by E. coli MTCC 739 cells treated with Kocuran-capped
silver glyconanoparticles. The CLSM images of biofilm formation in E. coli MTCC 739 (a) without treatment (control); (b) treated with
Kocuran-capped silver glyconanoparticles, 0.1 μg ml−1; (c) treated with Kocuran-capped silver glyconanoparticles, 1 μg ml−1; (d) treated with
Kocuran-capped silver glyconanoparticles, 5 μg ml−1; and (e) treated with Kocuran-capped silver glyconanoparticles, 10 μg ml−1. The E. coli
MTCC 739 cells were cultured in Muller–Hinton broth on Thermanox plastic cover slips at 37 °C for 24 h and then treated with 0.1, 1, 5 and
10 μg ml−1 of Kocuran-capped silver nanoparticles. The biofilm formation was observed by CLSM after staining with Live/Dead staining kit.
The untreated E. coli MTCC 739 represents the group that did not receive any treatment. The scale bars are 10 μm.

addition, the in vitro biofilm assay and confocal microscopic

Figure 9. Kocuran-capped silver glyconanoparticles coating inhibits
biofilm formation on silicone urethral catheters. The organisms were
cultured overnight with sterile silicone urethral catheters coated with
(+) and without (−) Kocuran-capped silver glyconanoparticles. The
biofilm formation by (a) S. aureus MTCC 96, (b) E. coli MTCC 739,
and (c) untreated catheter (control) after 24 h incubation was
demonstrated using crystal violet staining.
examination evidenced the efficacy of Kocuran-capped
AgNPs to inhibit the biofilm formation by S. aureus and
E. coli. The in vitro biocompatibility evaluation of the
Kocuran-capped silver glyconanoparticles on human gingival
fibroblasts demonstrated good cell proliferation as compared
to the commercial AgNPs suggesting that they are bio-
compatible and non-toxic in nature. Our data also suggests
that the Kocuran-capped AgNPs are hydrophilic, stable and
biodegradable nanomaterials which find application as coat-
ings on biomedical implants due to their intrinsic antibacterial
and antibiofilm properties and may help to prevent infections
and biofilm formation by drug-resistant pathogens as well as a
model system for studying various carbohydrate-carbohy-
drate, carbohydrate-protein and carbohydrate-cell
biointeractions.
Acknowledgments
The authors acknowledge the financial assistance provided to
Pombala Sujitha in the form of Senior Research Fellowship

13
Nanotechnology 25 (2014) 325101 C G Kumar and P Sujitha

Figure 10. Performance evaluation of Kocuran-capped silver glyconanoparticle-coating on silicone urethral catheters as a function of time.
The organisms were cultured overnight with sterile silicone urethral catheters coated with (+) and without (−) Kocuran-capped silver
glyconanoparticles. The biofilm formation by (a) S. aureus MTCC 96, and (b) E. coli MTCC 739 after 48, 72 and 96 h incubation was
demonstrated using crystal violet staining.

Figure 11. SEM images of Kocuran-capped silver glyconanoparticles coated urethral catheter. The surface was colonized with (a) S. aureus
MTCC 96 and (b) E. coli MTCC 739 after 48 h of incubation.

Figure 12. SEM images of uncoated catheter. The surface was colonized with (a) S. aureus MTCC 96 and (b) E. coli MTCC 739 after 48 h of
incubation.

14
Nanotechnology 25 (2014) 325101
Figure 13. In vitro biocompatibility studies of Kocuran-capped silver
glyconanoparticles on human gingival fibroblasts (HGF). The
cultured HGFs were treated with Kocuran, Kocuran-capped silver
glyconanoparticles and commercially available citrate-synthesized
AgNP at various concentrations (1–200 μg ml−1), incubated at 37 °C
for 48 h and measured for cell proliferation (%) using MTT assay.
by Council of Scientific and Industrial Research (CSIR), New
Delhi, India.
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