RESEARCH LETTER
Effect of silver nanoparticles and silver ions on growth and
adaptive response mechanisms of Pseudomonas putida mt-2
Nancy Hachicho, Philipp Hoffmann, Kristin Ahlert & Hermann J. Heipieper
Department of Environmental Biotechnology, Helmholtz Centre for Environmental Research – UFZ, Leipzig, Germany
Correspondence: Hermann J. Heipieper,
Department of Environmental Biotechnology,
UFZ Helmholtz Centre for Environmental
Research, Permoserstr. 15, 04318 Leipzig,
Germany.
Tel.: +49 341 2351694;
fax: +49 341 235451694;
e-mail: [email protected]
Received 21 February 2014; revised 30 April
2014; accepted 1 May 2014. Final version
published online 22 May 2014.
DOI: 10.1111/1574-6968.12460
Editor: Skorn Mongkolsuk
MICROBIOLOGY LETTERS
Keywords
silver nanoparticles; Pseudomonas putida;
toxicity; adaptation; cis-trans isomerization.
Abstract
The distribution and use of nanoparticles increased rapidly during the last
years, while the knowledge about mode of action, ecological tolerance and biodegradability of these chemicals is still insufficient. The effect of silver nanoparticles (AgNP) and free silver ions (Ag+, AgNO3) on Pseudomonas putida mt-2
as one of the best described bacterial strains for stress response were investigated. The effective concentration (EC50) causing 50% growth inhibition for
AgNP was about 250 mg L1, whereas this was only 0.175 mg L1 for AgNO3.
However, when calculating the amount of free silver ions released from AgNP
both tested compounds showed very similar results. Therefore, the antibacterial
activity of AgNP can be explained and reduced, respectively, to the amount of
silver ions released from the nanoparticles. Both tested compounds showed a
strong activation of the unique membrane adaptive response of Pseudomonas
strains, the cis-trans isomerization of unsaturated fatty acids, whereas another
important adaptive response of these bacteria, changes in cell surface hydrophobicity, measured as water contact angle, was not activated. These results are
important informations for the estimation of environmental tolerance of newly
developed, active ingredients like silver nanoparticles.
Introduction
Nanoparticles became an important and strong industry
since the last years. Many pharmaceuticals and care products of daily use are treated with these compounds. More
than 250 products worldwide were supplemented with silver nanoparticles in spring 2012 (Jiang et al., 2012). The
small size of nanoparticles between 1 and 100 nm
(Whitesides, 2003) enables these particles to cross several
borders allowing different applications as carrier molecules and antimicrobial agents (Kreuter, 2007; Arvizo
et al., 2012). One of the most promising fields of
nanotechnology is that of silver nanoparticles (AgNP).
The antibacterial activity of silver ions is well known and
has been used in ancient times where water was stored in
silver vessels to prevent their contamination by bacteria
(Silver, 2003). In combination with technical evolution,
the properties of silver are still of widespread interest and
new, silver-containing chemicals were designed and
synthesised. The distribution of AgNPs increased rapidly
during the last years, but the mode of action and the
FEMS Microbiol Lett 355 (2014) 71–77
ecological tolerance of these nano size particles are still
insufficient (Sondi & Salopek-Sondi, 2004; Greulich et al.,
2012).
The antibacterial activity of silver and the higher
toxicity against prokaryotic cells than eukaryotic cells are
the reasons for their intensive use in medicine. Wound
treatment with nanosilver supplemented dressing is
known to be extremely efficient for burns due to the
anti-inflammatory impact of silver (Atiyeh et al., 2007;
Chaloupka et al., 2010). Clinical equipment is covered
with antibacterial surfaces to diminish the risk for nosocomial infections (Chaloupka et al., 2010). The effective
mechanisms which causes the antibacterial effect of AgNPs are not been complete enlightened today. The release
of active silver ions from silver and AgNPs in aqueous
solutions has already been reported and is suspected to
attack the bacterial cell in multiple ways which causes
lethal cell damages (Morones et al., 2005; Lok et al.,
2006, 2007; Yang et al., 2009; Chaloupka et al., 2010).
The interactions of silver ions on different levels of the
bacterial cell are reported for cell wall components, the
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
72
cytoplasmic membrane, bacterial DNA as well as for
proteins and enzymes involved in energy household like
electron transport chain (Yamanaka et al., 2005; Jung
et al., 2008; Chaloupka et al., 2010). Proteomic studies of
Escherichia coli cells showed adaptive response against
silver ions by increasing the expression level of three
outer membrane protein precursors after treatment with
nanosilver to counteract the effect caused by interactions
of silver ions with the cell wall (Morones et al., 2005; Lok
et al., 2006; Pal et al., 2007). Whereas different routes for
synthesis are intensively investigated and improved, the
knowledge about waste treatment, biodegradability, toxicity and ecological tolerance of nanoparticles is still insufficient (Blaser et al., 2008; Fabrega et al., 2009). Thus, the
contamination of soils, sediments and groundwater with
hazardous chemicals represents an enormous problem for
environmental health. Especially in case of newly developed, active ingredients like silver nanoparticles, there is
the important question of environmental tolerance.
In the present study, the effect of silver nanoparticles
and free silver ions on growth and adaptive mechanisms
of one of the best investigated bacterial strains regarding
stress adaptation and response, Pseudomonas putida mt-2,
was compared.
Materials and methods
Chemicals
For all experiments, commercial silver nanoparticle
dispersion
(736503,
Silverjet
DGH-55LT-25C;
Sigma-Aldrich) was used. The chemical composition was
Ag (107.87 g mol1) to 50–60 wt. % in tetradecane.
According to manufacturer0 s product specification data
sheet, the particle size was < 10 nm. The dispersion was
1000-fold diluted in tetradecane, stored at 5 °C and treated with ultrasonic prior application. The influence of tetradecane in low concentrations up to 4.5 mM on
bacterial growth is negligible (data not shown). The use
as carbon source can be excluded due to the presence of
sodium succinate in sufficient quantities. All experiments
were additionally performed with silver nitrate AgNO3
(169.88 g mol1; ROTH) in aqueous solution instead of
AgNP solution. The AgNO3 concentrations were set in
accordance with the Ag+ concentrations of the AgNP
supplemented cultures.
N. Hachicho et al.
(Hartmans et al., 1989) with 4 g of Na2-succinate as carbon and energy source in 250 mL closed glass flasks. All
liquid cultures were incubated at 30 °C and 160 r.p.m. in
a gyratory shaker. Cell growth was monitored by optical
cell density measurement at 560 nm (OD560) using a
spectrophotometer. AgNP or AgNO3 were added in different concentrations to cells during the early exponential
growth phase. Cells were harvested in the late log phase
by centrifugation at 10 000 g for 15 min and washed five
times with KNO3 (10 mM, pH 7.0). The washed biomass
were resolved in 2 mL KNO3 and divided, wherein 1 mL
were stored at 5 °C for surface hydrophobicity analysis
and 1 mL were centrifuged again and the pellet was
stored at 20 °C for extraction of fatty acids.
Growth inhibition was measured by comparing the
differences in growth rate l [h1] [1] between stressed
cultures with that of control cultures (Heipieper et al.,
1995).
l½h1 ¼
ln xt2 ln xt1
:
t2 t1
(1)
In which xt2 was the optical density (OD560) at time t2,
xt1 was the optical density (OD560) at time t1, and t was
the incubation time in hours.
The growth inhibition lrel [%] of different concentrations of the stressor was defined as the percentage of
growth rates of cultures grown in presence of silver to
that of control cultures without addition of silver in the
form of silver AgNP or AgNO3. With µx as growth rate,
l [h1] of stressed culture and µc as growth rate l [h1]
of control which was set to 100 % (Heipieper et al.,
1995).
lrel ½% ¼
lx
100%
lc
(2)
Lipid extraction and FAME synthesis
The lipids were extracted from harvested biomass pellets
with chloroform/methanol/water as described by Bligh &
Dyer (1959). Fatty acid methyl esters (FAME) were prepared by incubation for 15 min at 95 °C in boron trifluoride/methanol applying the method of Morrison &
Smith (1964). FAMEs were extracted with hexane.
Analysis of fatty acid composition by GC-FID
Bacterial strain and culture conditions
Pure cultures of the bacterial strain Pseudomonas putida
mt-2 (DSM 6125) were used for growth inhibition
experiments. For growth inhibition experiments, bacterial
colonies were inoculated into 50 mL mineral salt media
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Analysis of FAME was performed using a quadruple GC
System (Agilent Technologies, 6890N Network GC System, 7683 Series Injector) equipped with a split/splitless
injector. A CP-Sil 88 capillary column (Chrompack, Middelburg, the Netherlands; length, 50 m; inner diameter,
FEMS Microbiol Lett 355 (2014) 71–77
73
Effect of silver nanoparticles on Pseudomonas putida mt-2
0.25 mm; 0.25 mm film) was used for the separation of
the FAME. The peak areas of the fatty acids in total ion
chromatograms (TIC) were used to determine their
relative amounts. The fatty acids were identified by
coinjection of authentic reference compounds obtained
from Supelco (Bellefonte). The relative amounts were
used to calculate the trans/cis ratio of unsaturated fatty
acids.
trans=cis ratio ¼
C16 : 1trans þ C18 : 1trans
C16 : 1cis þ C18 : 1cis
(3)
Preparation and measurement of surface
hydrophobicity
Washed biomass was added to 20 mL KNO3, and the
bacterial suspension was filtered through a membrane filter (0.45 lm, Ø 25 mm, cellulose nitrate, NC 45, Whatman) to create a bacterial-covered filter surface. The wet
filters were applied to bisected microscope slides prepared
with double-sided adhesive tape and dried for 2 h. Measurement was performed by video documentation of the
fall down of a water drop (3 lL volume, 40 lL s1 rate)
on the bacterial-covered filter surface using the drop
shape analyse system (DSA 100, Kr€
uss GmbH, Germany).
The contact angle h [°] between water/bacterial surface/
air was measured by image analysis of the recorded video
with the DSA software in the circle fitting mode. The procedure was performed four times per filter, and the average was calculated.
Results
Effect of AgNP and AgNO3 on bacterial growth
The effect of silver nanoparticles on growth and adaptive
response mechanisms of Pseudomonas putida mt-2 was
investigated and compared with the influence of silver
nitrate based on the silver content of ions and particles.
The stressor was added in the early exponential growth
phase, and cell growth was monitored by optical density
measurement. The relative growth rates lrel [%] were
plotted against the stressor concentration. The tolerance
of strain mt-2 against AgNP and AgNO3 are shown in
Fig. 1a and b. For both stressors, a dose-dependent
growth inhibition was investigated. The minimal inhibition concentrations (MIC) were 0.3 mg L1 for AgNO3
and 620 mg L1 for AgNP. The effective concentration
(EC50) causing 50% growth inhibition for AgNP was
about 250 mg L1, whereas this was only 0.175 mg L1
for AgNO3. By calculation of the estimated silver concentration based on the nanoparticle amount of 0.055% for
the 1000-fold diluted nanoparticle ink, a solid content
FEMS Microbiol Lett 355 (2014) 71–77
interpreted as silver concentration was used for the interpretation. So 240 mg L1 represent a real content of Ag+
of 0.055% (90.00055). This means 0.132 mg L1 and
1.22 lM, respectively, if the whole nanoparticle silver was
set free. By taking into consideration that about 1% of
the AgNP are present as Ag+ (Navarro et al., 2008), a
minimal silver concentration of 1.32 lg L1 or 0.012 lM
was estimated for free silver. Based on the molecular
weight of AgNO3 (MW 169.87 g mol1), the silver content was 63.5 %. The AgNO3 concentrations were set in
accordance with the calculated Ag+ concentrations of the
AgNP supplemented cultures.
The growth inhibition was also plotted against the silver content of the investigated chemicals (Fig. 2). The
figure allowed the direct comparison of growth inhibition
based on the silver content of AgNO3 and AgNP solution
regardless of their compound concentrations. The MIC
values were determined at Ag+ concentrations of
1.75 lM for AgNO3 supplemented cultures and 3.25 lM
for cells grown with AgNPs. The EC50 value was found
around 1 lM for both curves.
Effect of AgNP and AgNO3 on membrane fatty
acid composition and cell surface
hydrophobicity
The cells were harvested in the late exponential phase of
growth and analysed for their phospholipid fatty acid pattern and cell surface hydrophobicity to investigate and
compare the effect of AgNO3 and AgNP on physiological
properties of strain mt-2. From relative amounts of the
fatty acids, the trans/cis ratio of unsaturated fatty acids
was calculated. The presence of both stressors caused a
significant increase of the trans/cis ratio from 0.1 for control cultures to 0.85 for stressed cells (Fig. 1c and d). The
curve shape reflected the inhibitory effect of the added
substances.
The effect of AgNO3 and AgNPs on cell surface hydrophobicity of strain mt-2 was investigated using water contact angle measurements. The contact angles h (°) showed
no significant changes due to the addition of the stressors
in comparison with the control (Fig. 1e and f). The
values for h were between 30° and 40°.
Discussion
The growth inhibitory effects of AgNPs and AgNO3 on
strain Pseudomonas putida mt-2 showed that free silver
was about 1600 times more toxic than AgNP. The trend
of these results was confirmed by the observations of previous studies (Dibrov et al., 2002; Lee et al., 2003; Sondi
& Salopek-Sondi, 2004; Morones et al., 2005; Kim et al.,
2007; Pal et al., 2007; Park et al., 2009; Li et al., 2010).
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
74
N. Hachicho et al.
(b) 100
90
90
80
Growth rate (% of control)
Growth rate (% of control)
(a) 100
70
60
50
40
30
20
10
0
0.00
(e)
0.05
0.10
0.15
0.20
0.25
(d)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.05
0.10
0.15
0.20
0.25
(f)
40
30
20
0
100 200 300 400 500 600 700 800 900 1000
0
100 200 300 400 500 600 700 800 900 1000
0
100 200 300 400 500 600 700 800 900 1000
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
80
70
Contact angle (°)
70
Contact angle (°)
50
0.30
80
60
50
40
60
50
40
30
30
20
0.00
60
0
0.30
0.9
0.0
0.00
70
10
Trans/cis ratio of unsaturated fatty acids
Trans/cis ratio of unsaturated fatty acids
(c)
80
20
0.05
0.10
0.15
0.20
0.25
0.30
AgNP (mg L–1)
AgNO3 (mg L–1)
Fig. 1. Effect of AgNO3 (a, c, e) and AgNP (b, d, f) on relative growth rates (●, a, b), trans/cis ratio of unsaturated fatty acids (□, c, d) and water
contact angles (♦, e, f) of Pseudomonas putida mt-2.
However, the different formulations of AgNP and the
wide concentration range of the investigated chemicals
allow no direct comparison of previous results with those
obtained in this study. Most interestingly and relevant for
future ecotoxicological discussion is the fact that when
calculating the actual amount of free Ag+ ions released by
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
either AgNP or AgNO3, the toxic effect of both tested
compounds was very similar. Thus, the main toxic effect
of AgNP on P. putida was caused by the amount of Ag+
released into the culture medium. Earlier studies also
indicate a correlation between toxicity against bacteria
and emission of silver ions from nanoparticles (Morones
FEMS Microbiol Lett 355 (2014) 71–77
75
Effect of silver nanoparticles on Pseudomonas putida mt-2
100
Growth rate (% of control)
90
80
70
60
50
40
30
20
10
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Ag+ [µM]
Fig. 2. Effect of the actual concentrations of silver ions (Ag+)
released from AgNO3 (●) and AgNP (D) on relative growth rates of
Pseudomonas putida mt-2.
et al., 2005; Lok et al., 2007; Pal et al., 2007; Beer et al.,
2012). Other research groups observed higher toxicity for
AgNPs compared with AgNO3 in studies with the photoautotroph algae Chlamydomonas reinhardtii where interactions with cysteine lead the toxic effect increase
(Navarro et al., 2008). Other studies dealing with tolerance of human stem and blood cells compared with bacterial cells from Escherichia coli and Staphylococcus aureus
against silver in molecular form and as nanoparticles
observed no differences for their effective toxic concentrations (Greulich et al., 2012). Flow cytometric analysis of
eukaryotic lung cells exposed to AgNP suspensions and
AgNP supernatant showed that AgNPs did not influence
the toxicity of the suspension at high silver ion fractions
(Beer et al., 2012).
Both AgNO3 and AgNPs caused a strong increase in
the trans/cis ratio of unsaturated membrane fatty acids.
The isomerization of cis to trans unsaturated membrane
fatty acids is known to be a tool for the assessment of the
toxicity of membrane disturbing compounds (Heipieper
et al., 1995, 1996). Hereby, the degree of isomerization
directly depends on toxicity and concentration of membrane affecting agents. The purpose of the conversion of
the cis configuration to trans is apparently a rapid
decrease of the membrane fluidity to environmental factors causing an increase in membrane fluidity (Heipieper
et al., 1992, 2003, 2007). Therefore, the trans/cis ratio of
unsaturated fatty acids is an elegant, reliable and rapid
bioindicator for membrane stress in experimental setups
(Heipieper et al., 1995, 1996). Thus, the fact that both
Ag+ and AgNP have an impact on the trans/cis ratio of
unsaturated membrane fatty acids provides with a clear
evidence that their mode of action correlates with the
damage of the cell membrane and the collapse of their
function as a permeability barrier (Morones et al., 2005;
FEMS Microbiol Lett 355 (2014) 71–77
Li et al., 2010). An effective adaptation mechanism performed by Pseudomonas putida is the change in the state
of the form of the cis and trans-unsaturated fatty acids
which has already been reported for organic solvents and
heavy metals (Heipieper et al., 1996). Studies with Vibrio
cholerae demonstrated the massive damage of the membrane with collapse of the proton motive force induced
by low concentrations of free silver ions (Dibrov et al.,
2002). Main target for the toxic action of silver ions
might be membrane proteins leading to a disturbance of
general membrane integrity causing an increase in its
fluidity.
Another important adaptive mechanism that was intensely reported for Pseudomonas strains is their ability to
modify the hydrophobicity of their cell envelopes (Neumann et al., 2006; Baumgarten et al., 2012a, b). The
results show that neither the addition of AgNO3, nor that
of AgNPs led to significant changes in the contact angle
values of strain mt-2. Thus, the cell surfaces remain
hydrophilic at all tested concentrations. Accordingly,
changes in the surface properties as an adaptation mechanism to the toxic effects of AgNO3 and AgNPs can be
excluded.
It could be shown that the toxins induce a dose-dependent inhibition of growth of P. putida mt-2. While the
contact angle of the bacterial cells remained largely unaffected, the membrane composition is changing due to the
addition of AgNO3 and AgNPs. Pseudomonas putida mt-2
is able to use modifications of the trans/cis ratio to adapt
and counteract the changing conditions induced by addition of the toxins.
In addition to the Ag+ release by AgNP, one should
keep in mind for further studies that the toxicity of AgNPs could be also correlated with the particle size and
(Morones et al., 2005; Pal et al., 2007; Choi & Hu, 2008).
For this reason, studies with silver nanoparticles of different shape and size are of increasing interest. Only when
the mechanisms of action and the behaviour of silver
nanoparticles under various environmental conditions are
fully understood, it will be possible to achieve their full
potential. At the same time the risk assessment of AgNPs
should, despite the wide variety of applications, not be
neglected.
References
Arvizo RR, Bhattacharyya S, Kudgus RA, Giri K, Bhattacharya
R & Mukherjee P (2012) Intrinsic therapeutic applications
of noble metal nanoparticles: past, present and future. Chem
Soc Rev 41: 2943–2970.
Atiyeh BS, Costagliola M, Hayek SN & Dibo SA (2007) Effect
of silver on burn wound infection control and healing:
review of the literature. Burns 33: 139–148.
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
76
Baumgarten T, Sperling S, Seifert J, von Bergen M, Steiniger F,
Wick LY & Heipieper HJ (2012a) Membrane vesicle
formation as a multiple-stress response mechanism enhances
Pseudomonas putida DOT-T1E cell surface hydrophobicity
and biofilm formation. Appl Environ Microbiol 78: 6217–6224.
Baumgarten T, Vazquez J, Bastisch C, Veron W, Feuilloley
MGJ, Nietzsche S, Wick LY & Heipieper HJ (2012b)
Alkanols and chlorophenols cause different physiological
adaptive responses on the level of cell surface properties and
membrane vesicle formation in Pseudomonas putida
DOT-T1E. Appl Microbiol Biotechnol 93: 837–845.
Beer C, Foldbjerg R, Hayashi Y, Sutherland DS & Autrup H
(2012) Toxicity of silver nanoparticles—Nanoparticle or
silver ion? Toxicol Lett 208: 286–292.
Blaser SA, Scheringer M, MacLeod M & Hungerb€
uhler K
(2008) Estimation of cumulative aquatic exposure and risk
due to silver: contribution of nano-functionalized plastics
and textiles. Sci Total Environ 390: 396–409.
Bligh EG & Dyer WJ (1959) A rapid method of total lipid
extraction and purification. Can J Biochem Physiol 37:
911–917.
Chaloupka K, Malam Y & Seifalian AM (2010) Nanosilver as a
new generation of nanoproduct in biomedical applications.
Trends Biotechnol 28: 580–588.
Choi O & Hu Z (2008) Size dependent and reactive oxygen
species related nanosilver toxicity to nitrifying bacteria.
Environ Sci Technol 42: 4583–4588.
Dibrov P, Dzioba J, Gosink KK & H€ase CC (2002)
Chemiosmotic mechanism of antimicrobial activity of Ag+ in
Vibrio cholerae. Antimicrob Agents Chemother 46: 2668–2670.
Fabrega J, Renshaw JC & Lead JR (2009) Interactions of silver
nanoparticles with Pseudomonas putida biofilms. Environ Sci
Technol 43: 9004–9009.
Greulich C, Braun D, Peetsch A, Diendorf J, Siebers B, Epple
M & Koller M (2012) The toxic effect of silver ions and
silver nanoparticles towards bacteria and human cells occurs
in the same concentration range. RSC Adv 2: 6981–6987.
Hartmans S, Smits JP, van der Werf MJ, Volkering F & de
Bont JA (1989) Metabolism of styrene oxide and
2-phenylethanol in the styrene-degrading Xanthobacter
strain 124X. Appl Environ Microbiol 55: 2850–2855.
Heipieper HJ, Diefenbach R & Keweloh H (1992) Conversion
of cis-unsaturated fatty acids to trans, a possible mechanism
for the protection of phenol-degrading Pseudomonas putida
P8 from substrate toxicity. Appl Environ Microbiol 58: 1847–
1852.
Heipieper HJ, Loffeld B, Keweloh H & de Bont JAM (1995) The
cis/trans isomerization of unsaturated fatty acids in
Pseudomonas putida S12: an indicator for environmental stress
due to organic compounds. Chemosphere 30: 1041–1051.
Heipieper HJ, Meulenbeld G, van Oirschot Q & de Bont JAM
(1996) Effect of environmental factors on the trans/cis ratio
of unsaturated fatty acids in Pseudomonas putida S12. Appl
Environ Microbiol 62: 2773–2777.
Heipieper HJ, Meinhardt F & Segura A (2003) The cis-trans
isomerase of unsaturated fatty acids in Pseudomonas and
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
N. Hachicho et al.
Vibrio: biochemistry, molecular biology and physiological
function of a unique stress adaptive mechanism. FEMS
Microbiol Lett 229: 1–7.
Heipieper HJ, Neumann G, Cornelissen S & Meinhardt F
(2007) Solvent-tolerant bacteria for biotransformations in
two-phase fermentation systems. Appl Microbiol Biotechnol
74: 961–973.
Jiang HS, Li M, Chang F-Y, Li W & Yin L-Y (2012)
Physiological analysis of silver nanoparticles and AgNO3
toxicity to Spirodela polyrhiza. Environ Toxicol Chem 31:
1880–1886.
Jung WK, Koo HC, Kim KW, Shin S, Kim SH & Park YH
(2008) Antibacterial activity and mechanism of action of the
silver ion in Staphylococcus aureus and Escherichia coli. Appl
Environ Microbiol 74: 2171–2178.
Kim JS, Kuk E, Yu KN et al. (2007) Antimicrobial effects of
silver nanoparticles. Nanomed Nanotechnol Biol Med 3: 95–
101.
Kreuter J (2007) Nanoparticles—a historical perspective. Int J
Pharm 331: 1–10.
Lee HJ, Yeo SY & Jeong SH (2003) Antibacterial effect of
nanosized silver colloidal solution on textile fabrics. J Mater
Sci 38: 2199–2204.
Li WR, Xie XB, Shi QS, Zeng HY, Ou-Yang YS & Chen YB
(2010) Antibacterial activity and mechanism of silver
nanoparticles on Escherichia coli. Appl Microbiol Biotechnol
85: 1115–1122.
Lok CN, Ho CM, Chen R, He QY, Yu WY, Sun H, Tam PK,
Chiu JF & Che CM (2006) Proteomic analysis of the mode
of antibacterial action of silver nanoparticles. J Proteome Res
5: 916–924.
Lok C-N, Ho C-M, Chen R, He Q-Y, Yu W-Y, Sun H, Tam
P-H, Chiu J-F & Che C-M (2007) Silver nanoparticles:
partial oxidation and antibacterial activities. J Biol Inorg
Chem 12: 527–534.
Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB,
Ramırez JT & Yacaman MJ (2005) The bactericidal effect of
silver nanoparticles. Nanotechnology 16: 2346.
Morrison WR & Smith LM (1964) Preparation of fatty acid
methyl esters and dimethylacetals from lipids with boron
fluoride-methanol. J Lipid Res 5: 600–608.
Navarro E, Piccapietra F, Wagner B, Marconi F, Kaegi R,
Odzak N, Sigg L & Behra R (2008) Toxicity of silver
nanoparticles to Chlamydomonas reinhardtii. Environ Sci
Technol 42: 8959–8964.
Neumann G, Cornelissen S, van Breukelen F, Hunger S, Lippold
H, Loffhagen N, Wick LY & Heipieper HJ (2006) Energetics
and surface properties of Pseudomonas putida DOT-T1E in a
two-phase fermentation system with 1-decanol as second
phase. Appl Environ Microbiol 72: 4232–4238.
Pal S, Tak YK & Song JM (2007) Does the antibacterial
activity of silver nanoparticles depend on the shape of the
nanoparticle? a study of the Gram-negative bacterium
Escherichia coli. Appl Environ Microbiol 73: 1712–1720.
Park H-J, Kim JY, Kim J, Lee J-H, Hahn J-S, Gu MB & Yoon
J (2009) Silver-ion-mediated reactive oxygen species
FEMS Microbiol Lett 355 (2014) 71–77
Effect of silver nanoparticles on Pseudomonas putida mt-2
generation affecting bactericidal activity. Water Res 43:
1027–1032.
Silver S (2003) Bacterial silver resistance: molecular biology
and uses and misuses of silver compounds. FEMS Microbiol
Rev 27: 341–353.
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: 177–182.
Whitesides GM (2003) The ‘right’ size in nanobiotechnology.
Nat Biotechnol 21: 1161–1165.
FEMS Microbiol Lett 355 (2014) 71–77
77
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: 7589–
7593.
Yang W, Shen C, Ji Q, An H, Wang J, Liu Q & Zhang Z
(2009) Food storage material silver nanoparticles interfere
with DNA replication fidelity and bind with DNA.
Nanotechnology 20: 0957–4484.
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved