International Journal of Medical Microbiology 305 (2015) 85–95

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International Journal of Medical Microbiology

journal homepage: www.elsevier.com/locate/ijmm

Antibacterial activity of silver and zinc nanoparticles against Vibrio

cholerae and enterotoxic Escherichia coli
Wesam Salem a,b , Deborah R. Leitner a,1 , Franz G. Zingl a,1 , Gebhart Schratter c ,
Ruth Prassl c , Walter Goessler d , Joachim Reidl a , Stefan Schild a,∗
a
University of Graz, Institute of Molecular Biosciences, BioTechMed-Graz, Humboldtstrasse 50, A-8010 Graz, Austria
b
South Valley University, Faculty of Science, Qena, Egypt
c
Institute of Biophysics, Medical University of Graz, BioTechMed-Graz, Schmiedlstraße 6, 8042 Graz, Austria
d
Institute for Chemistry, Analytical Chemistry, University of Graz, BioTechMed-Graz, 8010 Graz, Austria

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

Article history: Vibrio cholerae and enterotoxic Escherichia coli (ETEC) remain two dominant bacterial causes of severe
Received 5 June 2014 secretory diarrhea and still a significant cause of death, especially in developing countries. In order to
Received in revised form 30 October 2014 investigate new effective and inexpensive therapeutic approaches, we analyzed nanoparticles synthe-
Accepted 4 November 2014
sized by a green approach using corresponding salt (silver or zinc nitrate) with aqueous extract of Caltropis
procera fruit or leaves. We characterized the quantity and quality of nanoparticles by UV–visible wave-
Keywords:
length scans and nanoparticle tracking analysis. Nanoparticles could be synthesized in reproducible yields
Caltropis procera
of approximately 108 particles/ml with mode particles sizes of approx. 90–100 nm. Antibacterial activity
Nanoparticles
In vivo
against two pathogens was assessed by minimal inhibitory concentration assays and survival curves. Both
Colonization pathogens exhibited similar resistance profiles with minimal inhibitory concentrations ranging between
Infant mouse model 5 × 105 and 107 particles/ml. Interestingly, zinc nanoparticles showed a slightly higher efficacy, but sub-
Biofilm lethal concentrations caused adverse effects and resulted in increased biofilm formation of V. cholerae.
Minimal inhibitory concentration Using the expression levels of the outer membrane porin OmpT as an indicator for cAMP levels, our results
Survival curve suggest that zinc nanoparticles inhibit adenylyl cyclase activity. This consequently deceases the levels
Therapeutic agent of this second messenger, which is a known inhibitor of biofilm formation. Finally, we demonstrated
Antimicrobial activity
that a single oral administration of silver nanoparticles to infant mice colonized with V. cholerae or ETEC
significantly reduces the colonization rates of the pathogens by 75- or 100-fold, respectively.
© 2014 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/3.0/).

Introduction ratio, which enable intimate interactions with microbial mem-

branes (Allaker, 2010; Morones et al., 2005). In addition, inorganic
Recently, nanotechnology has become increasingly important antibacterial agents such as metal and metal oxides are advanta-
in the biomedical and pharmaceutical areas as alternative antimi- geous compared to organic compound due to their stability (Sawai,
crobial strategy due to re-emergence infectious diseases and the 2003; Sondi and Sondi, 2004). Among these metal oxides, ZnO has
appearance of antibiotic-resistant strains especially within Gram- attracted a special attention as antibacterial agent. For instance,
negative microorganisms (Desselberger, 2000). Biosynthesis of ZnO inhibits the adhesion and internalization of enterotoxigenic
green nanoparticles using plant extracts is an interesting area in the E. coli (ETEC) into enterocytes (Roselli et al., 2003). In addition,
field of nanotechnology, which has economic and eco-friendly ben- ZnO nanoparticles (ZnO-NPs) exhibit antibacterial activity and can
efits over chemical and physical methods of synthesis (Suzan et al., reduce the attachment and viability of microbes on biomedical sur-
2014). Nanoparticles (NPs) are typically no greater than 100 nm faces (Brayner et al., 2006; Yamamoto, 2001). Interestingly, several
in size and their biocidal effectiveness is suggested to be owing results suggest a selective toxicity of ZnO-NPs preferentially tar-
to a combination of their small size and high surface-to-volume geting prokaryotic systems, although killing of cancer cells has
also been demonstrated (Hanley et al., 2008; Reddy et al., 2007;
Taccola et al., 2011). Several mechanisms have been reported for the
∗ Corresponding author. Tel.: +43 0316 380 1970; fax: +43 0316 380 9019. antibacterial activity of ZnO-NPs. For example ZnO-NPs can inter-
E-mail address: [email protected] (S. Schild). act with membrane lipids and disorganize the membrane structure,
1
These authors contributed equally to this work. which leads to loss of membrane integrity, malfunction, and finally

http://dx.doi.org/10.1016/j.ijmm.2014.11.005
1438-4221/© 2014 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).
86 W. Salem et al. / International Journal of Medical Microbiology 305 (2015) 85–95

to bacterial death (Krishnamoorthy et al., 2012; Zhang et al., 2007). while the reduction was considered to occur due to the phenolics,
ZnO may also penetrate into bacterial cells at a nanoscale level terpenoids, polysaccharides and flavonoids present in the extract
and result in the production of toxic oxygen radicals, which dam- (Babu and Prabu, 2011).
age DNA, cell membranes or cell proteins, and may finally lead to In the present study, we synthesized metallic ZnO- and Ag-NPs
the inhibition of bacterial growth and eventually to bacterial death using leaf and fruit extract of Calotropis procera and characterized
(Apperlot et al., 2009; Irzh et al., 2010; Makhulf et al., 2005; Moody their antibacterial activity against V. cholerae and ETEC. Espe-
and Hassan, 1982; Zhang et al., 2007). cially Ag-NPs synthesized from leaf extracts showed the most
Furthermore, Ag+ ions and Ag-based compounds are highly toxic robust antibacterial efficacy against both pathogens throughout the
to several microorganisms, which make them interesting candi- study. Furthermore, these Ag-NPs reduced fitness of the bacteria in
dates for multiple applications in the medical field (Furno et al., biofilms as well as in vivo.
2004; Prakash et al., 2013). Ag is generally used as nitrate salt, but in
the form of Ag nanoparticles (Ag-NPs) the surface area is increased
and thereby antimicrobial efficacy is greatly enhanced. Though Ag- Materials and methods
NPs find use in many antibacterial applications, the action of this
metal on microbes is not fully known. It has been hypothesized that Bacterial strains, culture conditions and supplements. V.
silver nanoparticles can cause cell lysis or growth inhibition via var- cholerae AC53 and ETEC H10407, spontaneous streptomycin-
ious mechanisms (Kim et al., 2007; Prabhu and Poulose, 2012). The resistant (SmR ) derivatives of the clinical isolates O1 El Tor
lethality of silver for bacteria can also be in part explained by thiol- Ogawa E7946 (Miller et al., 1989); (Schild et al., 2007) or ETEC
group reactions that inactivate enzymes (Chen and Schluesener, O78:H11:K80 (Evans and Evans, 1973), were used in this study.
2008; Feng et al., 2000). Also, Steuber and colleagues suggested Unless stated otherwise strains were grown in LB broth with aer-
a mechanism for Ag+ action in Vibrio alginolyticus involving the ation at 37 ◦ C or for biofilm formation under static conditions at
direct displacement of FAD from the holo-enzyme Na+ -NQR, which room temperature (RT). If required, streptomycin was used with a
results in loss of enzyme activity (Steuber et al., 1997). In summary, final concentration of 100 ␮g/ml.
silver treatment inhibits DNA replication, expression of ribosomal
and other cellular proteins, and interferes with the bacterial elec-
tron transport chain (Bragg and Rainnie, 1974; Feng et al., 2000; Plant materials and preparation of the extracts
Yamanaka et al., 2005).
Several reports demonstrated the synthesis of ZnO- and Ag-NPs Healthy leaves and fruits of Caltropis procera were collected from
from natural sources like plants or microorganisms by green chem- South Valley University campus at Qena city (Egypt), washed thor-
istry approaches (Babu and Prabu, 2011). The use of plant extracts oughly with tap water followed by distilled water, and air dried on
for nanoparticles synthesis may be advantageous over other biolog- a paper towel for 4–6 days. Dry leaves were shredded and ground
ical processes, because it drops the elaborate process of maintaining in a tissue grinder (IKA A10, Germany) to fine powder. Ten grams
cell cultures and can also be used for large-scale NPs synthesis of the powder were dissolved in 100 ml sterile double distilled
(Jeeva et al., 2014). Additionally, the green chemistry approach for water and heated for 1 h at 80 ◦ C. The obtained extract was filtered
the synthesis of NPs using plants avoids the generation of toxic through Rotilabo® Typ 601P filter paper; the filtrate was collected
byproducts. Among the various known synthesis methods, plant in a 250 ml Erlenmeyer flask and then stored at 4 ◦ C for further use
mediated NPs synthesis is preferred as it is cost-effective, eco- (modified from (Verastegui et al., 1996).
friendly and safe for human therapeutic use (Kumar and Yadav,
2009).
Diarrheal diseases are still a common worldwide cause of mor- Green synthesis of silver and zinc oxide nanoparticles (Ag-NPs and
bidity and mortality especially in the developing world. Within ZnO-NPs)
these areas, V. cholerae (∼25%) followed by ETEC (∼15%) are
most prevalent bacterial pathogens causing diarrheal diseases Ag-NPs and ZnO-NPs were essentially synthesized as previously
(Chowdhury et al., 2011; Walker et al., 2007). V. cholerae is the described (Babu and Prabu, 2011; Prakash et al., 2013; Sangeetha
causative agent of cholera, a life-threatening secretory diarrheal et al., 2011, 2012; Sun et al., 2014; Suzan et al., 2014; Vimala et al.,
disease. According to Southeastern and Central Asia reports the 2014) using leaves (L) or fruits (F) extracts from C. procera result-
annual acute diarrheal cases for V. cholerae infection were esti- ing in the four different types of nanoparticles Ag-NPs-L, Ag-NPs-F,
mated more than 1 million (WHO, 2013). ETEC is a common cause ZnO-NPs-L and ZnO-NPs-F. Solutions with silver nitrate or zinc
of traveler’s diarrhea, being responsible for up to one-half of diar- nitrate (without C. procera extract) were also incubated at the same
rheal episodes in travelers to Asia, Africa and Latin America (Gupta conditions and served as a negative control (Kumar et al., 2011).
et al., 2008; Qadri et al., 2005; Sanchez and Holmgren, 2005; Tobias To obtain Ag-NPs, 20 ml of a 1 mM AgNO3 (Sigma–Aldrich)
et al., 2011). Particularly children show a high mortality rate in solution were added drop-wise to 20 ml of the respective aque-
developing countries where diarrheal diseases remain the second ous plant extract of C. procera under constant stirring at 80 ◦ C
most common cause of death (Levine, 2006). Even today treatment within 30–45 min, for the reduction of Ag+ ions. This material
of these diarrheal diseases relies on a simple rehydration therapy, was incubated in the dark (to minimize the photoactivation of sil-
sometimes in combination with antimicrobial agents (Sack et al., ver nitrate) at 37 ◦ C. The synthesis of ZnO-NPs was performed as
2004). The rehydration therapy is highly effective, but appropriate previously described with some modifications. Briefly, 2 g of zinc
sterile solutions, antibiotics and medical expertise are not always nitrate (Sigma–Aldrich) was dissolved in 100 ml aqueous leaf or
available and during the explosive outbreaks medical facilities can- fruit extracts solution of C. procera under constant stirring. After
not cope with the massive numbers of incoming patients. Thus, complete dissolution of the mixture, the solution was kept under
alternative strategies should be investigated. vigorous stirring at 80 ◦ C for 2 h, subsequently allowed to cool at
Calotropis procera is a shrub (F: Asclepiadaceae) distributed in room temperature and the supernatant was discarded. Obtained
West Africa, Asia and other parts of the tropics. The plant is erect, NPs solutions were centrifuged at 4,500 rpm for 15 min after thor-
tall, large, branched and perennial with milky latex throughout ough washing and dried at 80 ◦ C for 7–8 h. Crude pellets were
(Irvine, 1961). Interestingly, Babu and Prabu recently described the then resuspended in sterile double distilled water, filtered through
Ag-NPs synthesis using aqueous extract of Calotropis procera flower, 0.2 ␮m filter and stored at 4 ◦ C in the dark prior to their use.
 W. Salem et al. / International Journal of Medical Microbiology 305 (2015) 85–95 87

UV–vis spectrophotometry growth was defined as the lowest concentration of NPs, which
inhibited bacterial growth. Growth was defined by an at least 2-
The reduction of Ag+ ions or ZnO was assessed by measuring the fold increase of the OD600 compared to the negative control (LB
UV–vis spectrum of 1 ml aliquots of sample in a cuvette as described only). To confirm bacterial growth inhibition and determine lack
earlier (Wiley et al., 2006). UV–vis spectral analysis for Ag-NPs and of metabolic activity, 40 ␮L of p-iodonitrotetrazolium violet INT
ZnO-NPs was carried out by measuring the optical density (OD) (0.2 mg/mL, Sigma–Aldrich) was added to microplate wells and
using the SPECTROstarNANO , BMG labtech, Germany scanning spec- reincubated at 37 ◦ C for 30 min (Eloff, 1998). The MIC in the INT
trophotometer. Measurements were performed between 220 and assay was defined as the lowest concentration of NPs that pre-
1,000 nm with a resolution of 1 nm. Silver nitrate (1 mM) or zinc vented color change as described earlier (Namrita et al., 2013).
nitrate (2%) were used as a blank, respectively.
Growth kinetics and viability
Inductively coupled plasma mass spectrometry (ICP-MS)
Growth kinetics were essentially performed as previously
Elements were determined in the samples after mineralization described in transparent 24-well plates (Greiner) with 1 ml culture
with nitric acid using ICP-MS. Briefly, the liquid samples (∼500 mg volume using LB broth, LB broth supplemented with leaf extract
weighed to 0.1 mg) were placed in 12 ml quartz vessels, 1 ml sub- (10%) or LB broth supplemented with fruit extract (10%) starting
boiled nitric acid was added and the samples were placed in the at an OD600 = 0.01 (Moisi et al., 2013; Seper et al., 2011). These
autoclave (UltraCLAVE IV, EMLS, Leutkirch, Germany). Then the final concentrations of the plant extracts are at least 2-fold higher
autoclave was pressurized with argon to 40 bars and the samples than the respective MIC of plant extracts with NPs. The OD600 was
heated in 45 min to a temperature of 250 ◦ C and kept at this tem- monitored every 30 min in the SPECTROstarNano microplate reader
perature for 45 min. After cooling the samples were transferred into (BMG Labtech) at 37 ◦ C with shaking. For presentation of data, at
15 ml polypropylene tubes (Greiner Bio-One). Zn (determined at least six independent growth curves were monitored for each strain
m/z 66) and Ag (determined at m/z 107) were determined with ICP- tested. The mean values were calculated and plotted. At 24 h via-
MS (Agilent 7500ce, Agilent Technologies, Waldbronn, Germany) bility in presence and absence of plant extracts was determined
after appropriate dilution. The accuracy of the results was vali- by plating appropriate dilutions of the cultures on LB plates. The
dated with the certified reference material 1640a (trace elements obtained CFU/ml from at least three independent measurements
in water, NIST, Gaithersburg, ML, USA). are presented as mean ± standard deviation.

Nanoparticle tracking analysis (NTA) measurements Static biofilm assay

To determine the size characteristics of the bio-synthesized Static biofilms were performed in microtiter plates by crystal
nanoparticles NTA was performed using the NanoSight LM10 HS- violet staining essentially as previously published (Seper et al.,
488FT14 instrument (Malvern Instruments, Herrenberg, Germany). 2011), with some modifications. Briefly, the respective strains were
The nanoparticle solutions were diluted in filter-sterilized grown over night on LB agar plates, suspended in LB, adjusted
(0.02 mm) double distilled water and 300 ␮l of each sample was to an OD600 of 0.02. 130 ␮l of this dilution were placed in a 96
injected to the viewing unit using a disposable syringe. The con- well microtiter plate (U bottom, Sterilin) for 24 h at RT. After
centration of the NPs was adjusted to a particle number between 24 h, 20 ␮l of Ag-NPs-L, Ag-NPs-F, ZnO-NPs-L or ZnO-NPs-F solu-
106 and 109 particles per ml. A laser (wavelength 405 nm) illumi- tions with concentrations of ∼ 108 NPs/ml were added. Addition
nates the particles from aside, and the particles act as point scatters of 20 ␮l of LB broth, the plant leaf or fruit extracts from C. pro-
moving under Brownian motion. Despite rapid movements of the cera served as control. After another 24 h incubation at RT, wells
particles, they can be tracked by a conventional CCD camera. The were subsequently rinsed with dH2 O using a microplate washer
recorded video can subsequently be analyzed analytically by a soft- (Anthos Mikrosysteme GmbH, Fluido2), biofilm was stained with
ware program (NTA 2.3, Build 0025). The samples were measured 0.1% crystal violet, solubilized in 96% ethanol and the OD595 was
for 60 s with manual shutter and gain adjustments. After capture measured (SPECTROstarNANO , BMG Labtech) to quantify the amount
of the diffusion coefficient and track lengths for the individual par- of biofilm.
ticles a quite accurate determination of the individual particle size
can be made (ASTM, 2012; Patrick et al., 2013). Particle concentra- Preparation of outer membrane proteins (OMPs) and whole-cell
tions of the original nanoparticle solutions were calculated by the lysates (WCL)
measured concentration of the diluted samples multiplied by the
dilution factor. OMPs were essentially prepared as previously published (Roier
et al., 2013). Briefly, ON cultures of V. cholerae or ETEC grown in
Determination of the minimum inhibitory concentration (MIC) by LB with or without sublethal concentrations of Ag-NPs-L or Ag-
growth and INT reduction assay NPs-F (both 1 × 106 NPs/ml) as well as ZnO-NPs-L or ZnO-NPs-F
(both 1 × 105 NPs/ml) respectively, as well as 0.025 mM of AgNO3
Overnight cultures of V. cholerae or ETEC were subcultured or 0.1 mM of Zn(NO3 )2 solution were harvested by centrifugation
1:10,000 into LB. Samples of 100 ␮l bacterial culture were placed (3,200 × g, 10 min, 4 ◦ C), washed once in HEPES buffer (10 mM,
into 96-well plates and 10 ␮l of appropriate serial dilutions of Ag- pH 7.4) and resuspended in 1 ml HEPES buffer (10 mM, pH 7.4).
NPs-L, Ag-NPs-F, ZnO-NPs-L or ZnO-NPs-F were added. At least two Then the suspension was transferred in a cryo-tube and cells were
independent preparations of each NP type were tested. Leaf or fruit disrupted by homogenization with 0.1 mm glass beads in combina-
extracts alone were also tested and showed no effects compared to tion with a PowerLyzerTM 24 (MO BIO Laboratories, Inc.), applying
LB broth. An additional control consisted of NPs-free supernatants three times, 1 min cycles at 3400 rpm with 1 min intervals on ice
from NPs solutions obtained after two consecutive centrifugation between each cycle. Unbroken cells were removed by centrifuga-
steps (20,000 rpm, 4 h, 4 ◦ C). The absence of NPs in the supernatant tion (15,600 × g, 2 min, 4 ◦ C). The supernatant containing the OMPs
was confirmed by NTA. After 16 h incubation in a humid cham- was transferred into a new tube and centrifuged again (15,600 × g,
ber at 37 ◦ C, the optical density (OD600 ) was measured using the 30 min, 4 ◦ C). The membrane pellet was re-suspended in 0.4 ml
SpectrostarNANO Microplate Reader (BMG Labtech). The MIC for HEPES buffer (10 mM, pH 7.4). To solubilize the cytoplasmic
88 W. Salem et al. / International Journal of Medical Microbiology 305 (2015) 85–95

membrane, 0.4 ml HEPES buffer (10 mM, pH 7.4) with 2% sarco- were determined by back-calculation to the original volume of the
syl was added and incubated at room temperature (RT) for 30 min. homogenized small intestine.
After centrifugation (15,600 × g, 30 min, 4 ◦ C), the pellet contain-
ing the OMPs was washed once with 0.5 ml HEPES buffer (10 mM, Statistical analysis
pH 7.4) and finally re-suspended in 50 ␮l HEPES buffer (10 mM, pH
7.4). Purified OMPs were stored at −20 ◦ C. Data were analyzed using the Mann–Whitney U test or a
The protein concentrations of OMP preparations were deter- Kruskal–Wallis test followed by post hoc Dunn’s multiple compar-
mined by photometric measurements of the absorbances at isons. Differences were considered significant at P values of ≤0.05.
260 nm and 280 nm using a Beckman Coulter DU730 spec- For all statistical analyses, GraphPad Prism version 4.0a was used.
trophotometer in combination with a TrayCell (Hellma)
and the Warburg–Christian equation given as mg pro-
Results
tein/ml = [(1.31 × A280) − (0.57 × A260)] × dilution factor
(Warburg and Christian, 1941).
Characterization of the nanoparticles
SDS-PAGE and immunoblot analysis
Zinc oxide and silver nanoparticles (ZnO-NPs and Ag-NPs) were
synthesized according to established protocols using leaf (L) and
To separate proteins the standard sodium dodecyl sulfate-
fruit extracts (F) from C. procera (Geethalakshmi and Sarada, 2010;
polyacrylamide gel electrophoresis (SDS-PAGE) procedure in
Hui et al., 2004; Sangeetha et al., 2011; Song and Kim, 2009),
combination with 15% gels and the Prestained Protein Marker Broad
resulting in the four different types of nanoparticles ZnO-NPs-L,
Range (New England Biolabs) as a molecular mass standard was
ZnO-NPs-F, Ag-NPs-L and Ag-NPs-F. After the addition of leaf and
used (Laemmli, 1970). Approximately 5 ␮g protein was loaded for
fruit extracts to the silver or zinc nitrate solutions, color changes
each sample. Proteins were stained according to Kang et al. (Kang
appeared within 30 min indicating the completion of the reaction,
et al., 2002) or transferred to a nitrocellulose membrane (Amer-
which is due to the excitation of plasmon vibrations in the metal
sham) for immunoblot analysis, which was essentially performed
nanoparticles (data not shown). In contrast, the control silver or
as described previously (Roier et al., 2012), using anti-OmpU or
zinc nitrate solution without extracts showed no color change (data
anti-OmpT antisera generated in mouse (1:500 diluted in 10%
not shown). The intensity of colors steadily increased along the
skim milk) as primary and peroxidase-conjugated goat anti-mouse
incubation period. Finally, Ag-NPs-L and Ag-NPs-F solutions exhib-
(diluted 1:10,000 in 10% skim milk, Dianova GmbH, Hamburg) as
ited a dark brown color, while solutions of Zn-NPs-L and Zn-NPs-F
secondary antibody, respectively.
exhibited dark yellow color. This may be due to the excitation of the
surface plasmon resonance (SPR) effect (Haes and Van Duyne, 2002)
Survival curve of V. cholerae and ETEC in the presence of Ag-NPs-L
and the reduction of either AgNO3 (Mulvaney et al., 1996) or zinc
nitrate (Sangeetha et al., 2011). The reduction of aqueous extracts
For the time-dependent survival analysis, a bacterial overnight
by silver or zinc ions and the formation of each NP-type were con-
culture was diluted 1:10,000 using LB-Sm broth supplemented
firmed using UV–vis spectroscopy (Fig. 1). A wavelength scans in
with aliquots of Ag-NPs-L (final concentration of 2.4 × 107 or
the UV–vis spectra revealed an absorption peak at approximately
1.2 × 107 NPs/ml). Cultures were grown without agitation at 37 ◦ C
 = 340 for ZnO-NPs-L and Zn-NPs-F (Fig. 1A and B). Furthermore
(using same conditions as for the MIC assay described above),
Ag-NPs-L and Ag-NPs-F exhibited characteristic absorption peaks
and 100 ␮l were collected at the indicated time intervals, serially
at approximately  = 370 nm as previously published (Jeeva et al.,
diluted in LB-Sm sterile broth and plated onto LB-Sm agar plates.
2014) (Fig. 1C and D). The presence of Zn and Ag in the NPs solutions
Viable colonies were counted after 16 h at 37 ◦ C. According to the
was confirmed by inductively coupled plasma mass spectrometry
volume plated on agar plates, the limit of detection for this assay
(ICP-MS), which revealed an at least 7-fold increase in case of Zn
was 10 CFU/mL.
or 400-fold in case of Ag in the NPs solutions compared to the
plant extracts, respectively (Table 1). The exact size distributions
In vivo colonization assay
and concentrations of independent NP preparations used in the
assays presented herein were determined by nanoparticle track-
In vivo experiments were performed as previously described
ing analysis (Fig. 2). Throughout the study, each type of NP has
with some modifactions (Leitner et al., 2013; Moisi et al., 2009;
been prepared at least three times without tremendous changes in
Schild et al., 2007). CD-1 mice (Charles River Laboratories) were
yield or quality, suggesting a reproducible production of the NPs.
used in all experiments in accordance with the rules of the ethics
In general, all NP preparations showed similar results with mean
committee at the University of Graz and the corresponding animal
concentrations ranging from 1.65 to 3.8 × 108 NPs/ml, mode par-
protocol, which has been approved by the Austrian Federal Ministry
ticle sizes of 88–100 nm and average particle size of 120–169 nm.
of Science and Research Ref. II/10b. Mice were housed with food and
water ad libitum and monitored under the care of full-time staff.
Mice were separated from their dams 1 h before infection. Sub- Table 1
sequently, they were anesthetized by inhalation of isoflurane gas Analysis of plant extracts as well as biosynthesized zinc and silver nanoparticles by
inductively coupled plasma mass spectrometry (ICP-MS).
and then inoculated by oral gavage with 50 ␮l of V. cholerae or ETEC
(approx. 1 × 105 CFU/mouse for both pathogens). To determine the Plant Total concentration Zn Total concentration Ag
exact inputs appropriate dilutions of the inocula were plated on extracts/nanoparticles (mg/kg) mean ± SDa (mg/kg) mean ± SD
LB-Sm plates. 6 h post-infection, infected mice were divided into Leaf extract 1.4 ± 0.1 <0.005
two groups. One group were treated orally with 50 ␮l of Ag-NPs–L Fruit extract 0.98 ± 0.14 <0.005
Zn-NPs-L 9.24 ± 0.54 n.d.b
(1.2 × 108 NPs/ml), while the other group received 50 ␮l saline solu-
Zn-NPs-F 25.6 ± 0.8 n.d.
tion. After 24 h, the mice were sacrificed and the small intestine Ag-NPs-L n.d. 2.1 ± 0.8
from each mouse was collected by dissection. The small intes- Ag-NPs-F n.d. 10 ± 3
tine was mechanically homogenized in LB broth with 15% glycerol a
Results are given as mean ± standard deviation of at least three independent
and appropriate 1:10 dilutions were plated on LB-Sm. After incu- preparations.
bation at 37 ◦ C ON, the colonization rates in CFU/small intestine b
Not determined.
 W. Salem et al. / International Journal of Medical Microbiology 305 (2015) 85–95 89

Fig. 1. UV–vis spectra for the reaction mixture containing of nanoparticles (NPs) synthesized from C. procera leaves (L) and fruits (F). Shown are the UV–vis absorption spectra
from 220 to 800 nm of ZnO-NPs-L(A), ZnO-NPs-F (B), Ag-NPs-L (C) and Ag-NPs-F (D).

The difference between mode and average particle sizes indicates

a non-parametric distribution of the NPs with the majority ranging
around 90–100 nm in size as well as a minor population with bigger
diameters. No aggregations or debris were detected by visualiza-
tion of the NPs within the nanoparticle tracking analysis (data not
shown), which indicates that NP suspensions are quite pure and
homogenous.

Antimicrobial activity of ZnO-NPs and Ag-NPs against V. cholerae

and ETEC.

Antibacterial activity of the different NPs synthesized by C. pro-

cera plant extracts against the human pathogens V. cholerae and
ETEC was analyzed by minimal inhibitory concentration (MIC) and
INT assays (Fig. 3). MIC was defined as the lowest concentration
at which no increase of the OD600 of the pathogens was observed.
However, in the absence of cell lysis the measurement of turbidity
cannot distinguish between live and dead bacteria (Carroll et al.,
2010). Thus, we additionally determined bacterial viability by a
colorimetric INT-formazan assay, which allows detection of viable
bacteria by their respiratory activity (Mann and Markham, 1998;
Palomino et al., 2002). All four NPs showed reproducible, effective
antibacterial activity with similar results in both assays (Fig. 3).
Fig. 2. Characterization of biosynthesized zinc and silver nanoparticles by nanopar- In general, ZnO-NPs showed a slightly higher efficacy compared
ticles tracking analysis (NTA). Shown are the concentrations of NPs (A) as well as the to Ag-NPs, with ZnO-NPs-F exhibiting the lowest MIC against both
NP sizes (B) presented as average (gray bars) and mode (white bars) NPs diameter
sizes in nm. At least three independent preparations of each NP type were analyzed.
pathogens. In detail, ZnO-NPs concentrations of 1.6 × 105 –1.2 × 106
The data is presented as the mean ± standard deviation (SD). per ml were sufficient for killing of V. cholerae and ETEC (Fig. 3A and
B), while in case of the Ag-NPs concentration of 5 × 106 –1.2 × 107
90 W. Salem et al. / International Journal of Medical Microbiology 305 (2015) 85–95

Fig. 3. Antibacterial activity of ZnO-Nps and Ag-Nps against V. cholerae and ETEC. Minimal inhibitory concentrations (MIC) for growth (gray bars) and metabolic activity
(open bars) against V. cholerae and ETEC were determined for ZnO-NPs-L (A), ZnO-NPs-F (B), Ag-NPs-L (C) and Ag-NPs-F (D). MIC for growth was determined by measuring
OD600 measurement and MIC for metabolic activity was assessed by a tetrazolium reduction assay. Please refer to the materials and methods section for details. Shown are
the medians from at least eight independent measurements. The error bars indicate the interquartile range.

per ml were necessary (Fig. 3C and D). Within the MIC and INT The pronounced increase in biofilm formation of V. cholerae
assays no detectable difference compared to the LB control were treated with ZnO-NPs was unexpected. However, it was recently
observed in the presence of plant extracts alone (data not shown). shown that adenylyl cyclases can be inhibited by Zn and conse-
Consistently, a comprehensive analysis revealed that neither the quently a sublethal concentration of ZnO-NPs could decrease cAMP
presence of leaf nor fruit extracts had a negative effect on the levels in V. cholerae (Klein et al., 2002, 2004). Besides others, the
growth kinetics or viability of V. cholerae or ETEC in comparison second messenger cAMP negatively effects biofilm formation as
to the LB control (Fig. 4). To exclude the possibility of soluble well as expression of the cholera toxin and the major coloniza-
side-products with antimicrobial activity in the NPs solution, the tion factor TCP in V. cholerae (Fong and Yildiz, 2008; Skorupski and
NPs-free supernatants from NPs solutions were obtained by cen- Taylor, 1997a, 1997b). Thus, treatment with sublethal concentra-
trifugation and tested in the MIC/INT assay. The absence of NPs tions of ZnO-NPs could cause adverse effects and induce biofilm
in the supernatant was confirmed by NTA. None of the NP-free formation and virulence. Interestingly, the outer membrane porin
supernatants derived from the four types of NPs solutions (ZnO- OmpT is positively regulated by this second messenger and lack
NPs-L, ZnO-NPs-F, Ag-NPs-L and Ag-NPs-F) exhibited any residual of cAMP completely abolishes OmpT expression (Li et al., 2002).
antimicrobial activity. Thus, the determined MIC correlates with In order to confirm the hypothesis of adenylyl cyclase inhibition
the presence of NPs. by ZnO-NPs, the outer membrane (OM) proteins were isolated
from V. cholerae cultures grown in absence or presence of sub-
ZnO-NPs and Ag-NPs have inverse effects on biofilm formation lethal concentrations of ZnO-NPs, Ag-NPs or zinc- and silver nitrate
solutions. Subsequently, these OM preparations were subjected to
Biofilm formation is an important survival strategy and persis- immunoblot analysis (Fig. 6). Kang staining and detection of OmpU
tence mode between epidemic outbreaks of the facultative human by immunoblot served as loading controls (Fig. 6A and C). While no
pathogens V. cholerae and ETEC (Ahmed et al., 2013; Colwell et al., difference in the abundance of OmpU was observed, all prepara-
2003). Recent findings suggest, that biofilms are a likely form in tions grown in presence of ZnO-NPs or zinc nitrate exhibited only
which V. cholerae and ETEC are taken up by humans, providing low levels of OmpT (Fig. 6B, lane 2, 3, 4). This result strengthens
a concentrated infective dose (Ahmed et al., 2013; Colwell et al., the hypothesis that treatment with ZnO-NPs inhibits the adenylyl
2003; Hall-Stoodley and Stoodley, 2005; Huo et al., 1996; Seper cyclase activity resulting in low cAMP levels.
et al., 2011; Tamayo et al., 2010). Since biofilms are generally known
to promote resistance against several antimicrobial agents (Lewis,
2005, 2008), we analyzed the impact of NPs on V. cholerae or ETEC Ag-NPs-L kills V. cholerae faster than ETEC
biofilms. Therefore, we allowed V. cholerae or ETEC to form static
biofilms for 24 h before ZnO-NPs-L, ZnO-NPs-F, Ag-NPs-L or Ag- Based on these results ZnO-NPs may interfere in cAMP signaling
NPs-F were added. Addition of LB broth, C. procera fruit or leaf and cause adverse effects. In addition, Ag-NPs-F had no impact on
extract served as controls, respectively. Finally, after an additional biofilm formation of both pathogens. Thus, Ag-NPs-L were selected
incubation period of 24 h the biofilm amount was quantified by for further analysis. First, the killing dynamic of Ag-NPs-L against
crystal violet staining (Fig. 5). Addition of plant extract (fruit or leaf) log-phase cultures of V. cholerae and ETEC was determined (Fig. 7).
had either no or a beneficial effect on biofilm formation in compari- Two different concentrations of Ag-NPs-L were tested, with one
son to the LB control. Thus, the NPs treated biofilms were compared close to the MIC and the other 2-fold higher. Addition of Ag-NPs-L at
with the appropriate fruit or leaf plant extract, respectively. Addi- these two different concentrations to a V. cholerae culture resulted
tion of Ag-NPs-F showed no change in biofilm formation of both in both cases in a rapid, continuous drop of viable cells with no
pathogens (Fig. 5A and B). In case of ETEC, treatment with ZnO- detectable CFUs after 2 h (Fig. 7A). In the case of ETEC, killing was
NPs-L, ZnO-NPs-F and Ag-NPs-L significantly reduced the biofilm delayed with a steady decrease of CFUs over time for both concen-
amount compared to the plant extract treated control, respectively trations tested. Finally no detection of viable cells could be observed
(Fig. 5B). This could also be observed for Ag-NPs-L treated biofilms after 20 h for the higher or 22 h for the lower concentration of Ag-
of V. cholerae (Fig. 5A). In contrast, treatment with ZnO-NPs-L and NPs-L, respectively (Fig. 7B). Based on these results, Ag-NPs-L kill V.
ZnO-NPs-F had an opposite effect and significantly increased the cholerae faster than ETEC. Furthermore, concentrations above the
biofilm amount of V. cholerae. MIC do not shorten the duration of killing.
 W. Salem et al. / International Journal of Medical Microbiology 305 (2015) 85–95 91

Fig. 5. Impact of NPs on biofilms of V. cholerae and ETEC. V. cholerae (A) and ETEC
(B) biofilms were allowed to grow for 24 h before ZnO-NPs-L, ZnO-NPs-F, Ag-NPs-L
or Ag-NPs-F were added. Addition of LB broth (LB), plant extracts from leaves (PE-L)
or fruits (PE-F) served as a control. After an additional 24 h biofilm formation was
quantified by crystal violet staining and subsequent determination of the OD595 .
Shown are the medians from at least eight independent measurements. The error
bars indicate the interquartile range. Significant differences between the data sets
are marked by asterisks (P < 0.05; Kruskal–Wallis test and post hoc Dunn’s multiple
comparisons).

Fig. 6. Immunoblot analysis of outer membrane preparations derived from V.

Fig. 4. C. procera plant extracts do not affect growth or viability of V. cholerae and cholerae treated with NPs. Depicted are the immunoblots for the detection of OmpU
ETEC. Shown are the growth kinetics of V. cholerae (A) and ETEC (B) in LB broth (A) or OmpT (B) as well as the protein profiles after Kang-staining (C) of OM
with leaf (dashed line) or fruit extracts (dotted line). LB broth alone served as con- preparations from V. cholerae grown in LB broth without supplements (lane 1) or
trol condition (solid line). Shown are the medians from at least six independent supplemented with zinc nitrate (lane 2), ZnO-NPs-L (lane 3), ZnO-NPs-F (lane 4), Ag-
measurements. (C) At 24 h the CFU/ml was determined by plating appropriate dilu- NPs-L (lane 5), Ag-NPs-F (lane 6), C. procera leaf extract (lane 7) or C. procera fruit
tions on LB plates. Data represent mean ± standard deviation (SD) of at least three extract (lane 8). Samples (approx. 6 ␮g protein each) were separated by SDS-PAGE
independent measurements. (15% gels) and protein bands were visualized according to Kang et al. (2002). Lines
to the left indicate the molecular masses of the protein standards in kDa.
92 W. Salem et al. / International Journal of Medical Microbiology 305 (2015) 85–95

Fig. 8. Impact of Ag-NPs-L on colonization fitness of V. cholerae and ETEC. Shown

are the recovered CFU per small intestine from mice orally infected with either V.
cholerae (A) or ETEC (B). 6 day old CD-1 mice were inoculated with either V. cholerae
or ETEC (approx. 1.5 × 106 CFU per mouse for both pathogens). At 6 h post infection
the control group (co) received 50 ␮l saline while treatment group received 50 ␮l Ag-
NPs-L solution (1.2 × 108 NPs/ml). At 24 h post infection, the small intestines were
collected, homogenized in LB medium and plated for CFU counting. Each column
represents the recovered CFU per small intestine from at least eight mice. The hori-
zontal bars indicate the median of each data set. Significant differences between the
data sets are marked by asterisks (P < 0.05; Kruskal–Wallis test and post hoc Dunn’s
multiple comparisons).
Fig. 7. Survival curve of V. cholerae and ETEC in presence of Ag-NPs-L. Shown are the
median CFU of V. cholerae (A) and ETEC (B) in LB broth supplemented with 2.4 × 107
Ag-NPs-L/ml (solid line) or 1.2 × 107 Ag-NPs-L/ml (dashed line) over time. Shown are
the medians from at least three independent measurements. The error bars indicate synthesized from leaf and fruit extracts of C. procera within 2 h
the interquartile range of each data set for each time point. The limit of detection of incubation. The main mechanism is based on a plant-assisted
for this assay was 10 CFU.
reduction due to C. procera phytochemicals including flavones,
organic acids, and quinones that are water soluble and are respon-
Oral treatment with Ag-NPS -L reduces colonization of V. cholerae sible for the immediate reduction of the ions (Iravani, 2011; Jha
and ETEC. et al., 2009; Prabhu and Poulose, 2012). The formation of polydis-
persed nanoparticles with concentrations of approx. 108 NPs/ml in
Finally, we addressed the impact of Ag-NPs-L on the small intes- the aqueous solution was confirmed by UV–vis spectrometry and
tine colonization of V. cholerae or ETEC using the infant mouse NTA analysis. The obtained yield, particle size and general quality
model. Therefore, mice were infected with V. cholerae or ETEC and is consistent with recent reports using aqueous extracts of neem
orally treated with an Ag-NPs-L solution 6 h post-infection. The and triphala leaves for synthesis of Ag-NPs or ZnO-NPs (Chandran
control group received an equal volume of saline buffer, which was et al., 2006; Ponarulselvam et al., 2012) (Asmita et al., 2012; Prabhu
also administered orally. The number of V. cholerae CFU recovered and Poulose, 2012; Sangeetha et al., 2011; Suzan et al., 2014; Vimala
from the small bowel at 24 h post-infection are shown in Fig. 8. All et al., 2014). It was also concluded that nanoparticle solutions were
control mice were stable colonized with median colonization lev- stable for more than six months with little signs of aggregation
els of 3 × 105 for V. cholerae or 5 × 104 CFU for ETEC, respectively. (Ankanna et al., 2010; Ponarulselvam et al., 2012; Saifuddin et al.,
Treatment with Ag-NPs-L resulted in a significant decrease in the 2009).
colonization levels, with a 50-fold reduction in case of V. cholerae The antibacterial activities of phyto-synthesized Ag-NPs and
and 200-fold reduction for ETEC compared to the control groups. ZnO-NPs were studied against the two Gram-negative pathogens V.
cholerae and ETEC, which are dominant bacterial causative agents
Discussion for diarrheal diseases. Although both NPs exhibited robust antibac-
terial activity in the MIC and INT assays, the ZnO-NPs generally
Several approaches have been employed to improve the meth- showed a slightly higher efficacy against both pathogens com-
ods for synthesizing Ag- and ZnO-NPs including chemical and pared to Ag-NPs. Interestingly, the amount of NPs necessary for
biological methods. Recently, nanoparticles synthesis based on growth inhibition (MIC assay) and inhibition of metabolic activ-
plant extracts is becoming more popular (Abasaheb et al., 2013; ity (INT assay) was almost equal, suggesting that the dominant
Suzan et al., 2014; Vimala et al., 2014). For example, Ag-NPs were antimicrobial target of the NPs are metabolic pathways of the bacte-
prepared from Aloe vera extract after 24 h of incubation or from ria. Noteworthy, antibiotic resistance of bacterial biofilms may be
Acalypha indica leaf extract after only 30 min of incubation using caused by poor antibiotic penetration within the biofilm matrix, an
aqueous solutions of silver nitrate (Chandran et al., 2006; Krishnaraj altered microenvironment or an adaptive bacterial response. Such
et al., 2010). In the current study, Ag- and ZnO-NPs were rapidly mechanisms acting together can raise the antibiotic resistance of
 W. Salem et al. / International Journal of Medical Microbiology 305 (2015) 85–95 93

biofilms by up to 1000 times in comparison with free living bacte- clarify any adverse effects and are necessary to support the safe use
rial cells (Mah and O’Toole, 2001). The aforementioned attributes of colloidal Ag-NPs. The fast and simple synthesis of nanoparticles
of biofilms place them amongst the most serious problems, which does not require special trained staff or expensive equipment and
medicine is currently facing. Thus, considerable effort has been is likely to be performed in epidemic areas. In contrast to antibi-
made to identify novel technologies that could form the basis of otics, prolonged exposure of bacteria to silver nanoparticles has not
anti-biofilm therapies, which are superior to current antibiotic resulted in the development of resistant cells so far. Silver nanopar-
treatment strategies. As a potential application for water disinfec- ticles as biocides tend to target multiple sites on or within bacterial
tion the NPs should also affect biofilms of these pathogens. cells and hence have a broad spectrum activity (Markowska et al.,
Interestingly, addition of ZnO-NPs to V. cholerae enhanced 2013). Thus, the application of silver nanoparticles as an effec-
biofilm formation. A possible explanation might be the inhibi- tive antimicrobial agent should not cause microbial resistance even
tion of the adenylyl cyclase by Zn-based compounds (Klein et al., after long-term usage.
2002, 2004). Consequently, cAMP levels are decreased, which de-
represses biofilm formation in V. cholerae. Consistent with this
Acknowledgements
hypothesis we found only low levels of the cAMP-induced porin
OmpT in the OM of V. cholerae treated with sublethal concen-
This work was supported by the Austrian FWF grants P25691 to
trations of ZnO-NPs. Noteworthy, the second messenger cAMP is
S. S., the W901-B12 (DK Molecular Enzymology) to D. R. L. and S.
involved in multiple regulatory pathways in V. cholerae including
S. as well as the Erasmus Mundus ELEMENT program funded with
natural competence, chitin colonization, outer membrane compo-
support of the European Commission. This publication reflects the
sition, motility, biofilm formation and virulence gene expression
view only of the author, and the Commission cannot be held respon-
(Antonova et al., 2012; Blokesch, 2012; Liang et al., 2007; Nielsen
sible for any use which may be made of the information contained
et al., 2010). Although in most cases the exact mechanisms are
therein.”
not yet fully understood, it was reported earlier that cAMP nega-
tively affects biofilm formation as well as expression of the cholera
toxin and the major colonization factor TCP (Fong and Yildiz, 2008; References
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