Research in Microbiology 162 (2011) 542e549

www.elsevier.com/locate/resmic

Antibacterial effect of silver nanoparticles on Staphylococcus aureus

Fateme Mirzajani a,1, Alireza Ghassempour a,*,1, Atousa Aliahmadi b,c, Mohammad Ali Esmaeili b
a
Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C. Evin, P.O.Box: 19835-389, Tehran, Iran
b
Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C. Evin, Tehran, Iran
c
Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran
Received 11 January 2011; accepted 26 March 2011
Available online 21 April 2011

Abstract

Antibacterial activity of silver nanoparticles (AgNPs) was investigated using Staphylococcus aureus PTCC1431 as a model of Gram-positive
bacteria. The mechanism of antibacterial activity of AgNPs was then studied by analyzing the growth, morphology, and molecular variations in
the cell wall. Experimental data showed that AgNPs at a concentration of 4 mg/ml completely inhibited bacterial growth. Transmission electron
microscopy results confirmed cell wall damage produced by AgNPs as well as accumulation of AgNPs in the bacterial membrane. Meanwhile,
the AgNP-treated bacteria were monitored by circular dichroism to reveal peptidoglycan variations. Some degree of variation in the a-helix
position of the peptide chain was observed. Moreover, increasing the AgNP concentration to 8 mg/ml resulted in release of muramic acid (MA)
into the medium, which could be attributed to cell wall distraction. A gas chromatography-tandem mass spectrometry analysis and release of
MA, as a bacterial indicator, showed that glycan strands may also be decomposed as a result of AgNP treatment.
Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Antibactericidal mechanism; Muramic acid; Peptidoglycan; Silver nanoparticles; Staphylococcus aureus

1. Introduction antibacterial activity (Shrivastava et al., 2007). AgNPs have

Nanotechnology merges materials at the atomic level with
unprecedented unique properties which can be suitably
manipulated for the desired application (Gleiter, 2000).
However, emerging literature on nanotoxicology has suggested
that nanoparticles might reveal new physicochemical and toxi-
cological properties (Lesniak et al., 2005; Handy et al., 2008;
Asharani et al., 2008). Colloidal silver nanoparticles (AgNPs)
smaller than 100 nm in at least one dimension have recently
received a great deal of attention and concern due to their
* Corresponding author. Department of Phytochemistry, Medicinal Plants
and Drugs Research Institute, Shahid Beheshti University, G.C. Evin, P.O.Box:
19835-389, Tehran, Iran.
E-mail addresses: f_mirzajnai@scientist.com, fateme.mirzajani@gmail.
com (F. Mirzajani), aghassempour@scientist.com, a-ghassempour@sbu.ac.ir
(A. Ghassempour), a_aliahmadi@sbu.ac.ir (A. Aliahmadi), m_esmaeili@sbu.
ac.ir (M.A. Esmaeili).
1
Tel.: þ98 21 22431598; fax: þ98 21 22431783.
been widely used for development of biological and pharma-
ceutical processes, products, and applications such as coating
material for medical devices, orthopedic or dental graft mate-
rials, topical aids for wound repair, clothing, underwear and
socks, textile products, and even washing machines (Chen and
Schluesener, 2008; Navarro et al., 2008).
Although it is well known that silver, whether in an ionic or
nanoparticle form, is highly toxic to microorganisms (Schreurs
and Rosenberg, 1982; Ghandour et al., 1988; Ahearn et al.,
1995; Feng et al., 2000; Dibrov et al., 2002), the mechanism
of its action has not been fully elucidated. In the case of Gram-
negative bacteria, a recent report demonstrated that AgNPs
make breaks through permeability of the outer membrane, and
these have been termed "pits", (Li et al., 2010). However, since
literature surveys did not conclude as to the mechanism of
AgNPs in Gram-positive bacteria, it is important to study this
effect.
It is postulated that primary cell wall structure, i.e. each of
the glycan strands and/or peptide branches, may be affected by

0923-2508/$ - see front matter Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.resmic.2011.04.009
 F. Mirzajani et al. / Research in Microbiology 162 (2011) 542e549
AgNPs. Peptidoglycan (PGN) is a specific and essential
element in the cell wall of almost all bacteria. Its main
structural features are linear glycan strands cross-linked by
short peptides. The glycan strands are made up of alternating
N-acetylglucosamine and N-acetylmuramic acid residues
linked by b-1/4 bonds (Vollmer et al., 2008) and the peptide
chain is composed of L-alanine, D-glutamine, L-lysine and D-
alanine in the case of Staphylococcus aureus. Although mur-
amic acid (MA) is the essential biomarker used to indicate
bacterial biomasses, (Zhang and Amelung, 1996), its amount
in cell walls of Gram-positives is 95%, while in Gram-
negative bacteria does not exceed 5% of the cell wall
weight. In the present study, it is hypothesized that release of
MA into the culture medium, followed by AgNP treatment,
can indicate their reactions with the cell wall PGN and its
destruction via glycan strands. Moreover, MA as a unique
biomarker, (Larsson and Saraf, 1997; Bal et al., 2002; Liang
et al., 2009) makes more accurate the study of cell wall
destruction followed by AgNP treatment.
2. Materials and methods
2.1. Materials
S. aureus PTCC1431 was purchased from the Iranian
Research Organization for Science and Technology (IROST,
Iran). HPLC grade methanol and ethanol were obtained from
Caledon Co. (Georgetown, Canada). In all procedures, Milli-Q
HPLC grade water was used. Ammonium acetate, silver nitrate,
3-sodium citrate, hydrochloric acid, myo-inositol, acetic
anhydride, potassium hydroxide, hydroxylamine hydrochlo-
ride, dichloromethane, DMSO, ethyl acetate, and hexane were
purchased from Merck. (Darmstadt, Germany). 4-dimethyl
amino pyridine was obtained from Alfa Aesar Co. (Karlsruhe,
Germany). MA and S. aureus cell wall PGN which were
supplied by SigmaeAldrich Co. (Steinheim, Germany) were
produced in Israel and Canada, respectively. Osmium tetroxide,
glutaraldehyde, d-10-camphor sulfonic acid (ammonium salt)
and polyvinyl alcohol, (PVA) were obtained from Acros
Organics Co. (Geel, Belgium).
2.2. Preparation and characterization of AgNPs
To prepare AgNP colloid, AgNO3 (1.0  103 M) aqueous
solution containing 0.1% PVA as a surfactant was reduced using
a dropwise addition of sodium citrate solution at near the boiling
point, (97  C  0.5). After 20 min, a yellowish gray suspension
was obtained. The resulting nanoparticles were purified by
centrifugation at 18,000 rpm  4 and washing with deionized
water. The optical properties of AgNPs were evaluated using
a 2501PC Shimadzu Co. (Kyoto, Japan) UVeVis spectropho-
tometer. In order to study their shape and morphology, EM 900
TEM from Zeiss Co. (Zeiss, Germany) was employed. Size of
the prepared AgNPs was studied using a Nanophox DLS
equipped with 632.8 nm HeNe-laser from Sympatec Co.
(Clausthal-Zellerfeld, Germany). The size measurement was
accomplished with respect to the UV absorption coefficient (e)
543
of the suspension at 632.8 nm and its refractive index (RI). For
X-ray powder diffraction study, the powder microcrystalline
sample was loaded into an aluminum sample holder that was
rotated during data collection to improve particle statistics and
to minimize preferred orientation effects. Diffraction data were
collected in the range of 1e80, (2q), on an STOE STADI P
diffractometer equipped with a scintillation detector, secondary
monochromator and Cu Ka1 radiation (l ¼ 1.5406 Å).
2.3. Cultivation, assay for minimum inhibitory
concentration (MIC) and minimal bactericidal
concencentration (MBC)
Aerobic cultivation was performed in Mueller-Hinton
culture media at 37  C under shaking at 450 rpm. Growth
curves of S. aureus exposed and unexposed to AgNPs were
determined based on the absorbing value of OD600. There was
no specific antibacterial guideline for nanoparticles and as
a result, the antistaphylococcal effect of AgNPs was evaluated
by microbroth dilution methods according to standard methods
recommended by the NCCLS (National Committee of Clinical
Laboratory Standards, 2005; Murray et al., 2007), with some
modifications to determine the minimum concentration of
each antibacterial agent required for inhibition (MIC) or
killing (MBC) of the test microorganism. The inoculant of S.
aureus was prepared from freshly cultured bacteria that were
adjusted to 0.5 McFarland standard turbidity using normal
saline and then further diluted (1:100) by sterile Mueller-
Hinton broth just before adding to the trays. AgNPs have an
absorbance in the range of 300e600 nm, so 0.5 McFarland
standard turbidity was used as well as OD600. AgNP serial
dilutions were made in a concentration range of 0.5e256 mg/
ml in sterile 96-well plastic microdilution trays containing
Mueller-Hinton broth. MICs were recorded after incubation
for 22 h at 37  C. Minimum bactericidal concentrations were
determined by subculturing 100 ml from each negative well
and from the positive growth control onto a nutrient agar plate.
MBCs were defined as the lowest concentration that could kill
99.9% of the test strains. All tests were performed in triplicate.
2.4. Transmission electron microscopy (TEM) study of
the bacteria
Electron micrographs of natural and 24h AgNP-treated
bacteria were taken using a Zeiss EM 900 transmission elec-
tron microscope (Zeiss, Germany). Cells of S. aureus were
collected by centrifugation at 10,000 rpm for 5 min and
resuspended in water. Subsequently, the samples were incu-
bated at 37  C for an hour. The cells were collected by
centrifugation (10000 rpm, 5 min) to remove water. To fix the
cells, 2.5% glutaraldehyde was then added and samples were
incubated at 4  C for 1 h. Afterward, cells were collected by
centrifugation (10,000 rpm, 5 min) and washed three times
with 0.1 M phosphate buffer. For final fixing of cells, 1%
osmium tetraoxide was added and samples were held at 25  C
for 1 h. Thereafter, samples were dehydrated with ethanol
544 F. Mirzajani et al. / Research in Microbiology 162 (2011) 542e549

solutions embedded in resin and held to polymerize; then,
a diamond knife was used for TEM sample preparation.
2.5. Bacterial cell wall (PGN) study
To study cell wall PGN, 1 l of MHB was inoculated by
a single colony of S. aureus was cultivated. The culture was
incubated at 37  0.5  C with shaking at 450 rpm. With respect
to MIC and OD600 results, the sample was treated with AgNPs
for 24 h. The sample was centrifuged at 12,000 rpm and filtered
through 0.22 mm. The supernatant was freeze-dried at 50  C
immediately and kept at 30  C for further analysis.
2.5.1. MA derivation and gas chromatography-tandem
mass spectrometry (GC-MSn)
A derivated bacterial culture medium was used for gas
chromatography-tandem mass spectrometry. In order to deri-
vate, the previously reported method was performed, (Zhang
and Amelung, 1996). In brief, the bacteria were treated with
AGNPs for 24 h. Biomass of the resulting suspension and the
control (without any treatment) were centrifuged (for 20 min
at 9000 rpm), filtered through 0.22 mm sterilized filter syringe,
dried under reduced pressure and kept at 4  C until analysis.
For evaluating variations, culture medium derivation was
studied using a Varian CP-3800 gas chromatograph equipped
with a 4000 MS ion-trap mass spectrometer. For this purpose,
the VF-1ms (CP8924), 30 m  0.25 mm ID (0.25 mm) column
was utilized in a split ratio of 1:30. The temperature program
was set as follows: the initial column temperature of 90  C
was held for 1 min, which was then increased to 250  C at
a rate of 5  C/min. Thereafter, the temperature was increased
again at a rate of 10  C/min to 270  C and finally held at this
temperature for 5 min.
2.5.2. Circular dichroism (CD) study of PGN
In order to study the influence of AgNPs on cell walls, the
standard suspension of S. aureus PGN was used. Homoge-
nized suspension of standard PGN was prepared on DMSO
using 30 min sonication and treated with AgNP solution at
a ratio of 1:1. The secondary structural changes in the samples
were evaluated by CD spectroscopy. Results of measurements
were recorded over a wavelength range of 200e260 nm using
an AVIV spectropolarimeter, model 215 (USA) with a 0.1 cm
path length sample cell. Calibration of the instrument was

Fig. 1. UV absorbance spectra in the range of 200e500 nm (a), dynamic light scattering and size distribution at 632.8 nm (b), TEM image (c) and X-ray powder
diffraction patterns (d-bottom) of AgNP colloidal aqueous. The Ag pattern from the JCPDS database is also shown for comparison (d-top).
 F. Mirzajani et al. / Research in Microbiology 162 (2011) 542e549
performed using aqueous solutions of d-10-camphor sulfonic
acid (ammonium salt) prior to use. All CD measurements were
carried out at 25  C with the help of a thermostatically
controlled cell holder. Spectra was collected at 1 nm intervals,
with a scan speed of 20 nm/min. Each spectrum was the
average of two scans and the noise in the data was smoothed
using AVIV 215 software (cdnn).
3. Results and discussions
The smaller AgNPs demonstrated stronger antibacterial
effects, (Choi and hu, 2008). Within these dimensions, AgNPs
are individually made up by the researchers and the suspension
is freshly used. The relative contributions from radiation
damping through resonant scattering and absorption strongly
depend on the particle size. However, particles smaller than
20 nm exhibit absorption only below 430 nm and light
extinction of particles larger than about 50 nm is dominated by
resonant scattering, (David et al., 2005). As can be seen from
the UV spectra in Fig. 1a, colloidal AgNPs have maximum
absorbance (lmax) at 426 nm. DLS analysis (Fig. 1b) of
synthesized AgNPs demonstrates that their size, volume mean
diameter (VMD), and surface mean diameter (SMD) were
18.34 nm (X99), 4.10 nm and 2.26 nm, respectively. Further-
more, the homogeneity of their size was evaluated within the
range of 0.1e1000 nm.
Based on the TEM image (1c), spherical AgNPs with
relatively uniform size distribution were obtained. The nano-
crystalline AgNPs were also identified in X-ray powder
diffraction analyses. For the AgNPs prepared with PVA as
a protective agent in aqueous solution, a typical X-ray powder
diffraction pattern is shown in Fig. 1d, which matches well
with the pattern for Ag (fcc) in the JCPDS reference library,
(cards 4-0783). Four distinct diffraction peaks were observed
at 2q, including 37.6 , 45.1 , 63.8 and 76.9 , respectively,
corresponding to the (111), (200), (220) and (311) crystalline
planes of cubic Ag. The average particle size estimated from
the DebeyeeScherer equation (He et al., 2001, 2002) is 17 nm,
which is comparable to results obtained from statistical anal-
ysis of the DLS. This supports the notion that Ag nanoparticles
are highly crystalline, with crystal grain sizes (detectable by
X-ray powder diffraction) similar to particle sizes (measurable
by DLS in colloid aqueous sample).
It is well known that Ag ions and Ag-based compounds
have a strong antibacterial effect. In addition, the bactericidal
effect of nanoparticles is dependent on the concentration of
nanoparticles and the initial bacterial concentration. Anti-
staphylococcal properties of AgNPs were confirmed according
to microbroth dilution susceptibility tests according to stan-
dard methods recommended by NCCLS. MIC and MBC
observed for synthetic nanoparticles were 2 and 4 mg/ml,
respectively (Mirzajani et al., 2010).
Generally, the antibacterial mechanism of chemical agents
depends on specific binding with the surface and the metabolism
of agents in the microorganism, especially PGN in the case of
Gram-positives. Moreover, in recent reports on Escherichia coli,
AgNPs start the process of pit formation and leakage of cellular
545
materials to cell culture (Li et al., 2010). This is followed by
inhibition of respiratory chain dehydrogenases and growth of
cells. Meanwhile, some proteins and phosphate lipids may affect
and induce a collapse of the membrane, cell decomposition and
eventually death. It was previously mentioned that AgNPs have
a specific interaction with amino acids (Li et al., 2004).
Furthermore, there are some reports on an AgNP mechanism for
nitrifying bacteria and also P. aeruginosa as a consequence of
their higher antibacterial potency toward Gram-negative than
Gram-positive bacteria. However, although there exist some
reports on the mechanism of Agþ on S. aureus as a Gram-
positive model bacteria, (Feng et al., 2000; Jung, et al., 2008),
there exists no detailed documentation on its mechanism of
action.
In order to group together all previously mentioned
mechanisms between AgNPs and bacterial cell walls, and to
reach conclusions pertaining to Gram-positive bacteria, anal-
yses were performed. These included study of the glycan and
peptide parts as well as overall bacterial fate. Thus, to evaluate
the influence of AgNPs on the bacterial PGN cell wall, and
specifically its peptide part, their CD spectra were recorded in
DMSO. CD spectra (Fig. 2) demonstrate the peptide-bond
absorption region (200e260 nm) of the treated PGNs. The
sample variations before and after AgNP treatment for 30 min
and 3 h show that AgNPs can influence the secondary struc-
tures of PGNs. In the region of 210e220 nm, a noticeable
change is observed. Previously, reports claimed that no vari-
ations in the secondary structures of proteins were observed as
a consequence of interaction with Agþ ions or binding with
AgNPs (Fayaz et al., 2010). Fig. 2 demonstrates that the
hydrogen bond and a-helix, i.e. the peptide secondary struc-
ture, is affected by AgNPs. Monitoring the association of PGN
solution and AgNPs after 30 min and 3 h demonstrated
significant changes occurring during the first 30 min of
treatment. Moreover, the study indicates that AgNPs change
the total configuration of PGN (Fig. 2).
TEM images of treated S. aureus are shown in Fig. 3. The
dark and light areas show where the sample had high and low
Fig. 2. CD spectra of PGN (— —), PGN treated with AgNP after 30 min
($$$$$) and 3 h (——).
546 F. Mirzajani et al. / Research in Microbiology 162 (2011) 542e549

electron density, respectively. Images of samples treated with
AgNPs were substantially different from those of untreated
cells (Fig. 3b). PGN fragmentation after treatment (Fig. 3b)
demonstrated their interaction with AgNPs, which caused cell
wall destruction in S. aureus.
As mentioned previously, it is hypothesized that the influ-
ence of AgNPs on bacteria can be monitored via each of the
affected parts. Their interaction with peptide branches was
investigated based on CD analysis. Study of AgNP effects on
cell wall PGN glycan strands indicated that the amount of MA
increased in culture media. GC-MSn was carried out before
and after treatment.
Fig. 4 is comparison between the control, treated culture
media and standard solution of MA. In the AgNP-treated
sample, myo-inositol was added to culture media prior to
derivation as an internal standard. It can be seen from the
Fig. 4c that AgNP treatment caused an increase in the MA
concentration in the culture medium. In addition, the presence
of MA in culture media was monitored via selected ion
monitoring (SIM at 212 m/z) of one of its major peaks (data

Fig. 3. Transmission electron microscopy images of S. aureus before (a) and after 24 h (b) AgNP treatment.
 F. Mirzajani et al. / Research in Microbiology 162 (2011) 542e549 547

Fig. 4. GCeMS/MS analysis of (a) standard solution, (b) culture media without treatment, (c) AgNP-treated culture media.

not included). MA release was also demonstrated by its
detection in standard spiked sample solution. From GC-MSn
results, it can be concluded that the structure of the cell wall is
damaged by glycan strands as well as peptide branches.
AgNPs caused fragmentation of glycan strands and released
small amounts of amino sugars and MA into culture media.
It was previously suggested that the bactericidal properties
of AgNPs lie in diverse mechanisms (Sondi and B. Salopek-
Sondi, 2004; Yamanaka et al., 2005; Vaidyanathan et al.,
2010; Li et al., 2010). As part of this research, Agþ antibac-
terial features were studied and mechanisms proposed. PGN
peptide branches may be influenced by AgNPs from each
amino acid or the final configuration. Fig. 2 demonstrates that
AgNPs not only changed the secondary structure (a-helix) of
the bacterial cell wall, but also destroyed its primary structure.
Many studies have focussed on the free sugar content of the
bacteria. Indeed, PGN glycan strands consist of amino sugars.
The AgNP interaction with glycan strands was studied based

Fig. 5. The proposed mechanism of AgNP and bacterial cell wall (a) interaction, (b) pit formation, Agþ release and PGN decomposition.
548 F. Mirzajani et al. / Research in Microbiology 162 (2011) 542e549
on increases in concentrations of MA as the bacterial indicator
in culture media. As shown by GC-MSn results, fragmentation
of glycan strands by AgNPs was one cause of bacterial
demolition. In addition to TEM images (Fig. 3b) showing
distortion of the S. aureus cell wall, GC-MSn results confirmed
this phenomenon concerning the increase in MA concentration
in the presence of AgNPs.
Overall, the literature indicates that there is a strong inter-
action between AgNPs and PGN. AgNPs interact with bacterial
cell walls individually or via Agþ release (Fig. 5a). Due to their
nano size, they make a connection with the cell wall and
generate “pits” on it (Fig. 5b). Thereafter, AgNPs accumulate
and begin to more strongly connect with underlying layers,
releasing Agþ as well. These phenomena more strongly influ-
ence the destruction of Gram-positive bacteria than Gram-
negative bacteria because they have a thicker PGN layer. They
are followed by variations in the conformation of a peptide’s
portion as well as glycan strands. Then, the grits of amino
sugars, like MA, were released into the culture media and the
engendered pits become larger. Finally, the uniform cell wall is
destroyed, and the bacterial organisms are vented outward.
In conclusion, TEM results showed destruction of the
Gram-positive bacterial cell wall PGN after AgNP treatment.
Moreover, the CD study indicated that the peptide portion of
PGN which was treated by AgNPs underwent structural
variation, as did the glycan strands. GC/MSn provided more
evidence of PGN glycan strand destruction and release of the
MA into the culture medium. With respect to all information
provided, AgNPs may make a connection with both sides of
the peptide and glycan ports of a bacterial cell wall and
generate}pits}on it. AgNPs attach to b-1/4 bonds of N-ace-
tylglucosamine and N-acetylMA of glycan strands, destroy
their bond and release them into the media. In conclusion, as
previously reported concerning Gram-negative bacteria, and
based on our evidence concerning Gram-positives, it was
confirmed that membrane pit formation may be an important
factor inhibiting bacterial growth. However, it remains
a mystery as to whether damage occurs on the lipopolysac-
charide layer of Gram-negatives or the peptidoglycan
membrane of Gram-positives.
Acknowledgments
Financial support from the Research Council of Shahid
Beheshti University and the Iran Nanotechnology Initiative
Council is gratefully acknowledged. This publication repre-
sents a component of the doctoral thesis of Fateme Mirzajani
at the Medicinal Plants and Drug Research Institute of Shahid
Beheshti University, Tehran, Iran.
References
Ahearn, D.G., May., L.L., Gabriel, M.M., 1995. Adherence of organisms to
silver-coated surfaces. J. Ind. Microbiol. 15, 372e376.
Asharani, P.V., Wu, Y.L., Gong, Z., Valiyaveettil, S., 2008. Toxicity of silver
nanoparticles in zebrafish models. Nanotechnology 19, 255102 (8 pp).
Bal, K., Larsson, L., Mielniczuk, E., Mielniczuk, Z., 2002. Structure of
muramic acid TMS derivative mass spectrum’s base ion (m/z¼185) used
for quantification of bacterial peptidoglycan. J. Microbiol. Meth. 48,
267e270.
Chen, X., Schluesener, H.J., 2008. Nanosilver: a nanoproduct in medical
application. Toxicol. Lett 176, 1e12.
Choi, O., Hu, Z., 2008. Size dependent and reactive oxygen species related
nanosilver toxicity to nitrifying bacteria. Environ. Sci. Technol. 42,
4583e4588.
David, D., Evanoff Jr., , Chumanov, G., 2005. Synthesis and optical properties
of silver nanoparticles and arrays. Chem. Phys. Chem. 6, 1221e1231.
Dibrov, P., Dzioba, J., Gosink, K.K., Häse, C.C., 2002. Chemiosmotic
mechanism of antimicrobial activity of Agþ in Vibrio cholerae. Anti-
microb. Agents Chemother. 46, 2668e2670.
Fayaz, A.M., Balaji, K., Girilal, M., Yadav, R., Kalaichelvan, P.T.,
Venketesan, R., 2010. Biogenic synthesis of silver nanoparticles and their
synergistic effect with antibiotics: a study against Gram-positive and Gram-
negative bacteria. Nanomedicine: Nanotechnol. Biol. Med. 6, 103e109.
Feng, Q.L., Wu, J., Chen, G.Q., Cui, F.Z., Kim, T.N., Kim, J.O., 2000. A
mechanistic study of the antibacterial effect of silverions on Escherichia
coli and Staphylococcus aureus. J. Biomed. Mater. Res. 52, 662e668.
Ghandour, W., Hubbard, J.A., Deistung, J., Hughes, M.N., Poole, R.K., 1988.
The uptake of silver ions by Escherichia coli K12: toxic effects and
interaction with copper ion. Appl. Microbiol. Biotechnol. 28, 559e565.
Gleiter, H., 2000. Nanostructured materials, basic concepts and micro-
structured. Acta Mater. 48, 1e12.
Handy, R.D., von der Kammer, F., Lead, J.R., Hassellöv, M., Owen, R.,
Crane, M., 2008. The ecotoxicology and chemistry of manufactured
nanoparticles. Ecotoxicology 17, 287e314.
He, R., Qian, X., Yin, J., Zhu, Z., 2002. Preparation of polychrome silver
nanoparticles in different solvents. J. Mater. Chem. 12, 3783e3786.
He, S., Yao, J., Xie, S., Gao, H., Pang, S., 2001. Superlattices of silver
nanoparticles passivated by mercaptan. J. Phys. D: Appl. Phys. 34,
3425e3429.
Jung, W.K., Koo, H.C., Kim, K.W., Shin, S., Kim, S.H., Park, Y.H., 2008.
Antibacterial activity and mechanism of action of the silver ion in
Staphylococcus aureus and Escherichia coli. Appl. Env. Microbiol. 74 (7),
2171e2178.
Larsson, L., Saraf, A., 1997. Use of gas chromatography-ion trap tandem mass
spectrometry for the detection and Characterization of microorganisms in
Complex samples. Molec. Biotech. 7, 279e287.
Lesniak, W., Bielinska, A.U., Sun, K., Janczak, K.W., Shi, X., Baker Jr., J.R.,
Balogh, L.P., 2005. Silver/Dendrimer Nanocomposites as biomarkers:
Fabrication, Characterization, in Vitro toxicity, and Intracellular detection.
Nano Let 11, 2123e2130.
Liang, C., Read, H.W., Balser, T.C., 2009. Reliability of muramic acid as
a bacterial biomarker is influenced by Methodological Artifacts from
Streptomycin. Microb. Ecol. 57, 494e500.
Li, T., Park, H.G., Lee, H.S., Choi, S.H., 2004. Circular dichroism study of
chiral biomolecules conjugated with silver nanoparticles. Nanotechnology
15, S660eS663.
Li, W.R., Xie, X.B., Shi, Q.S., Zeng, H.Y., OU-Yang, Y.S., Chen, Y.B., 2010.
Antibacterial activity and mechanism of silver nanoparticleson Escherichia
coli. Appl. Microbiol. Biotechnol. 85, 1115e1122.
Mirzajani, F., Ghassempour, A., Aliahmad, A., Esmaeili, M.A., 2010. Silver
Nanoparticle - Peptidoglycan Cell Wall Interaction. Proceedings of the
31st European Peptide Symposium, European Peptide Society, 2010.
Murray, P.R., Baron, E.J., Jorgensen, J.H., Landry, M.L., Pfaller, M.A.,
2007. Manual of Clinical Microbiology, nine ed. ASM press,
Washington.
National Committee of Clinical Laboratory Standards, 2005. Methods for Dilu-
tion Antimicrobial Susceptibility Test for Bacteria that Grow Aerobically.
National Committee of Clinical Laboratory Standards, Villanova, Penn.
Navarro, E., Piccapietra, F., Wagner, B., Marconi, F., Kaegir, R., Odzak, N.,
Sigg, L., Behra, R., 2008. Toxicity of silver nanoparticles to Chlamydo-
monas reinhardtii. Environ. Sci. Technol. 42, 8959e8964.
Schreurs, W.J.A., Rosenberg, H., 1982. Effect of silver ions on transport and
retention of phosphate by Escherichia coli. J. Bacteriol. 152, 7e13.
 F. Mirzajani et al. / Research in Microbiology 162 (2011) 542e549
Shrivastava, S., Bera, T., Roy, A., Singh, G., Ramachandrarao, P., Dash, D.,
2007. Characterization of enhanced antimicrobial effects of novel silver
nanoparticles. Nanotechnology 18, 9. 225103.
Sondi, I., Salopek-Sondi, B., 2004. Silver nanoparticles as antimicrobial agent:
a case study on E. coli as a model for Gram-negative bacteria. J. Colloid
Interface. Sci. 275, 177e182.
Vaidyanathan, R., Gopalram, S., Kalishwaralal, K., Deepak, V., Ram Kumar
Pandian, S., Gurunathan, S., 2010. Enhanced silver nanoparticle synthesis
by optimization of nitrate reductase activity. Colloid Surf. B. 75, 335e341.
549
Vollmer, W., Blanot, D., de Pedro, M.A., 2008. Peptidoglycan structure and
architecture. FEMS Microbiol. Rev. 32, 149e167.
Yamanaka, M., Hara, K., Kudo, J., 2005. Bactericidal actions of a silver ion
solution on Escherichia coli, studied by Energy-Filtering transmission
electron microscopy and Proteomic analysis. Appl. Environ. Microbiol. 71
(11), 7589e7593.
Zhang, X., Amelung, W., 1996. Gas chromatography determination of mur-
amic acid, glucosamine, mannosamine and galactosamine in soils. Soil
Boil. Biochem. 28, 1201e1206.