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ORIGINAL RESEARCH
published: 26 July 2018
doi: 10.3389/fphar.2018.00816
Edited by:
Gregory Franklin,
Institute of Plant Genetics (PAN),
Poland
Reviewed by:
Xing-Feng Zheng,
Second Military Medical University,
China
Karthik Siram,
PSG College of Pharmacy, India
Letizia Angiolella,
Sapienza Università di Roma, Italy
*Correspondence:
Aleksandra Królicka
aleksandra.krolicka@biotech.ug.edu.pl
Specialty section:
This article was submitted to
Ethnopharmacology,
a section of the journal
Frontiers in Pharmacology
Received: 26 March 2018
Accepted: 09 July 2018
Published: 26 July 2018
Citation:
Krychowiak M, Kawiak A,
Narajczyk M, Borowik A and
Królicka A (2018) Silver Nanoparticles
Combined With Naphthoquinones as
an Effective Synergistic Strategy
Against Staphylococcus aureus.
Front. Pharmacol. 9:816.
doi: 10.3389/fphar.2018.00816
Silver Nanoparticles Combined With
Naphthoquinones as an Effective
Synergistic Strategy Against
Staphylococcus aureus
Marta Krychowiak1, Anna Kawiak2, Magdalena Narajczyk3, Agnieszka Borowik4and
Aleksandra Królicka1*
1Laboratory of Biologically Active Compounds, Intercollegiate Faculty of Biotechnology, Medical University of Gda´
nsk,
University of Gda ´
nsk, Gda ´
nsk, Poland, 2Laboratory of Plant Protection and Biotechnology, Intercollegiate Faculty
of Biotechnology UG and MUG, University of Gda ´
nsk, Gda ´
nsk, Poland, 3Laboratory of Electron Microscopy, Faculty
of Biology, University of Gda´
nsk, Gda ´
nsk, Poland, 4Laboratory of Biophysics, Intercollegiate Faculty of Biotechnology UG
and MUG, University of Gda ´
nsk, Gda ´
nsk, Poland
Staphylococcus aureus is a human pathogen responsible for many antibiotic-resistant
infections, for instance burn wound infections, which pose a threat to human life.
Exploring possible synergy between various antimicrobial agents, like nanoparticles
and plant natural products, may provide new weapons to combat antibiotic resistant
pathogens. The objective of this study was to examine the potential of silver
nanoparticles (AgNPs) to enhance the antimicrobial activity of selected naphthoquinones
(NQs): plumbagin (PL), ramentaceone (RAM), droserone (DR), and 3-chloroplumbagin
(3ChPL). We also attempted to elucidate the mechanism by which the AgNPs enhance
the antimicrobial activity of NQs. We analyzed the interaction of AgNPs with bacterial
membrane and its effect on membrane stability (TEM analysis, staining with SYTO9
and propidium iodide), as well as aggregation of NQs on the surface of nanoparticles
(UV-Vis spectroscopy and DLS analysis). Our results demonstrated clearly a synergistic
activity of AgNPs and three out of four tested NQs (FBC indexes ≤0.375). This resulted
in an increase in their combined bactericidal effect toward the S. aureus reference
strain and the clinical isolates, which varied in resistance profiles. The synergistic effect
(FBC index = 0.375) resulting from combining 3ChPL with silver nitrate used as a
control, emphasized the role of the ionic form of silver released from nanoparticles in
their bactericidal activity in combination with NQs. The role of membrane damage and
AgNPs-NQ interactions in the observed synergy of silver nanoparticles and NQs was
also confirmed. Moreover, the described approach, based on the synergistic interaction
between the above mentioned agents enables a reduction of their effective doses, thus
significantly reducing cytotoxic effect of NQs toward eukaryotic HaCaT cells. Therefore,
the present study on the use of a combination of agents (AgNPs-NQs) suggests its
potential use as a possible strategy to combat antibiotic-resistant S. aureus.
Keywords: combination of antimicrobials, Droseraceae, plant secondary metabolite, 1,4-naphthoquinone,
antibiotic resistance, membrane damage, silver ions
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Krychowiak et al. Anti-staphylococcal Naphthoquinones and Silver Nanoparticles
INTRODUCTION
The effort placed on the exploration of new anti-infectious
approaches is justified in the near post-antibiotic era that
we face (Ventola, 2015). Drug resistance is observed in a
growing number of clinical and environmental isolates of bacteria
and fungi. Moreover, the problem of increasing antibiotic
resistance concerns most of antibiotic drugs approved to treat
infectious diseases (Magiorakos et al., 2012). Staphylococcus
aureus is a Gram-positive bacterium belonging to a group
of the most troublesome antibiotic resistant pathogens, so-
called “ESCAPE” according to Peterson (2009). Most infections
caused by S. aureus are associated with strains resistant to
β-lactams (and also other classes of antibiotics) and most
of them are healthcare-acquired like burn wound infections
(Tong et al., 2015). The likelihood of anti-infectious treatment
failing due to the antibiotic resistance is an increasingly
recurring problem. In order to address those issues, there
is an urgent need for the development of new, alternative
approaches.
Nanotechnology is a fast-developing field which can be
applied to diverse medical issues. Silver nanoparticles (AgNPs),
a typical example of nanomaterials, are widely studied because
of their antimicrobial activity (Rai et al., 2009). Different
formulations containing AgNPs have been proposed so far as
antimicrobial treatments for burn wound infections (Jain et al.,
2009), to improve wound healing (Tian et al., 2007), control
implant infections (Juan et al., 2010), or to avoid medical
device-related infections (Roe et al., 2008). The mechanism of
antimicrobial action of AgNPs is complex and depends on both
nanoparticles and silver ions released from their surface, and
involves an interaction with many cellular components (Dakal
et al., 2016). Many natural compounds of plant origin also possess
antimicrobial activity and have a potential to fight antibiotic-
resistant pathogens (Phoenix et al., 2014). In our previous
study we demonstrated synergistic activity of an extract from
Drosera binata when combined with AgNPs toward S. aureus
(Krychowiak et al., 2014). Naphthoquinones (NQs), a group
of secondary metabolites with a naphthalene backbone, were
found to be the most prevalent and active constituents among all
secondary metabolites detected in the extract studied. Droserone,
3-chloroplumbagin, plumbagin, and its isomer ramentaceone
(Figure 1) are the most prevalent naphthoquinones synthesized
in tissues of plants of the Droseraceae family (Juniper et al.,
1989;Kreher et al., 1990;Gaascht et al., 2013;Krychowiak
et al., 2014). The antibacterial activity of most of NQs
concerns mainly Gram-positive bacteria like S. aureus, as most
of Gram-negative bacteria are intrinsically resistant to NQs
(Riffel et al., 2002;Krolicka et al., 2008, 2009;Moreira et al.,
2017).
Although the direct use of naphthoquinones as antibacterial
agents is limited due to their cytotoxicity toward the eukaryotic
cells (Babich and Stern, 1993), their potential as alternative
antimicrobials in an era of growing antibiotic resistance is
worth exploring. Thus, we employed an approach based on
a combination of antimicrobials to verify the possible use of
NQs as anti-staphylococcal agents. Our study aims to examine
AgNPs as agents that enhance bactericidal potential of NQs and
in consequence reduce the effective dose of these secondary
metabolites. Moreover, to investigate the possible mechanism of
the observed synergistic effect, we also verified the hypothesis of
the mode of synergistic action of AgNPs and NQs based on cell
membrane damage caused by nanoparticles and their interaction
with naphthoquinones.
MATERIALS AND METHODS
Bacterial Strains and Antimicrobial
Agents
In this study we used one reference strain of S. aureus (ATTC
25923) and four strains isolated from patients (Laboratory of
Microbiology at the Provincial Hospital in Gda´
nsk, Poland),
with different antibiotic resistance profile established according
to CLSI guidelines (CLSI, 2012) for oxacillin, vancomycin and
ciprofloxacin (Supplementary Table 1). The clinical isolates
were described as follows: 703 k (oxacillin-resistant), 614 k
(oxacillin- and ciprofloxacin-resistant), 56/AS (vancomycin-
and ciprofloxacin-resistant), 6347 (oxacillin-, vancomycin-, and
ciprofloxacin-resistant). All bacterial strains used in this study
are stored in the Laboratory of Biologically Active Compounds,
Department of Biotechnology, IFB UG and MUG Gda´
nsk,
Poland.
Vancomycin, ciprofloxacin, oxacillin and plumbagin (PL)
were purchased from Sigma-Aldrich and Daptomycin (DAP)
from Selleck Chemicals. Protegrin-1 (PR) was synthesized
by Lipopharm Sp. z o.o. (Poland). Ramentaceone (RAM)
was obtained from the University of Pretoria, Republic of
South Africa. Droserone (DR) and 3-chloroplumbagin (3ChPL)
were synthesized at the Technical University of Gda´
nsk by E.
Paluszkiewicz, Ph.D., as described by Krychowiak et al. (2014).
FIGURE 1 | Chemical structure of four naphthoquinones selected for this
study, present in tissues of carnivorous plants. Plumbagin: R1= CH3, R2= H,
R3= H; 3-chloroplumbagin: R1= CH3, R2= Cl, R3= H; droserone:
R1= CH3, R2= OH, R3= H; ramentaceone: R1= H, R2= H, R3= CH3.
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Krychowiak et al. Anti-staphylococcal Naphthoquinones and Silver Nanoparticles
AgNPs solutions were purchased from Prochimia Surfaces Sp.
z o.o. (Poland). As described by the manufacturer, AgNPs
are water soluble, spherical nanostructures coated with
(11-Mercaptoundecyl)-N,N,N-trimethylammonium chloride,
characterized by the average size of 5.5 nm and dispersity
level of 15%. The initial concentration of AgNPs solutions was
6.74 ×1014 NPs/ml equivalent to 615 µg/ml (SPR maximum,
λmax: 420–424 nm).
Antibacterial Assays
To characterize antibacterial potential of all agents tested in
this study, alone and in combinations, we determined their
minimal bactericidal concentration (MBC), which is the lowest
concentration of agent that after 24 h reduces the number of
bacterial cells in the initial inoculum by 99.9% (3 logarithms). To
determine the MBC we used the Microdilutions Broth Method
(Thornsberry, 1991) according to the CLSI guidelines (CLSI,
1996). To that effect, we prepared twofold dilutions of the tested
agents in 96-well plates, with final volume of 0.1 mL per well.
The tested concentrations ranged from 0.125 to 512 µg/mL.
Bacterial inoculum was prepared from late-log cultures (37◦C,
150 rpm) in Cation-adjusted Mueller-Hinton Broth (CA-MHB)
prepared from colonies on BHI agar plates (24 h, 37◦C).
Liquid culture was diluted in fresh CA-MHB medium to
0.5 McF (according to McFarland standards) measured with
a densitometer (DensiMeter II, EMO, Brno), corresponding
to ∼1.5 ×108CFU/mL (colony forming units per mL). The
bacterial suspension was then diluted with medium to a density of
2.5–5 ×106CFU and 10 µL aliquots were allocated to each well
of the 96-well plates. In addition, dilutions containing untreated
bacteria were plated out to check the number of bacterial cells in
wells in each experiment. The 96-well plates were then incubated
without shaking for 24 h at 37◦C. Wells with no visible growth
of bacteria were plated out on BHI agar plates and incubated
for 24 h at 37◦C, colony counts made to determine the lowest
bactericidal concentration of each agent, or combination of
agents. Each concentration or combination of concentrations was
tested in triplicate and each experiment was performed at least in
triplicate.
Synergy Testing
In order to examine the interaction mechanism of tested
combinations of antimicrobials (synergistic, additive or
antagonistic) we employed the Checkerboard Titration Method
(Thornsberry, 1991). This method is based on testing the
combination of two agents at their concentration gradient
obtained by twofold dilutions. The range of the tested
concentrations of each agent was from 0.03×MBC to 2×
MBC. Such an approach allowed determination of changes
(reduction or increase) in the minimal effective concentrations
of the analyzed agents after their combination. The mode
of action of two agents used simultaneously is expressed
mathematically as the Fractional Bactericidal Concentration
index (FBC index), calculated according to the equation: FBC
index = FBCA/MBCA+FBCB/MBCB, where MBCAand
MBCBare the lowest bactericidal concentrations of A and
B tested separately, whereas FBCAand FBCBare the lowest
bactericidal concentrations of agent A and B when they are
used in combination. The nature of interaction of two agents
is described by the value of FBC index: lower or equal to 0.5 –
synergistic interaction, higher than 0.5 but lower or equal to 1 –
additive interaction, higher than 1 – antagonistic interaction.
The isobole method (Barenbaum, 1978) was used to visualize the
character of interaction between AgNPs and NQs whereby the
isobole curve being a line connecting the points of the lowest
bactericidal concentrations of the combined agents.
Time-Dependent Killing
In each step of time-dependent killing assay, the bacterial cells
were cultured aerobically in CA-MHB medium at 37◦C and
150 rpm. Bacterial cells grown overnight were diluted 100 times
in fresh medium and cultured for another 4 h (37◦C, 150 rpm)
until they reached a mid-log phase. Then, bacterial suspension
was diluted in a fresh medium to 0.5 McF measured with a
densitometer (DensiMeter II, EMO, Brno), 0.1 mL aliquots of
inoculum were then transferred to wells on 24-well plate with
1 mL of medium containing the tested antimicrobials (8 µg/mL
3ChPL or 3.6 µg/mL AgNPs) and their combinations (8 µg/mL
3ChPL combined with 3.6 µg/mL AgNPs). Wells containing
medium without antimicrobials were treated as control. Each
well was sampled (50 µL) for colony counts at the following
timepoints: 0, 1, 2, 4, 6, and 24 h and the plates were incubated
as described above. Each concentration of agent, or combination
of agents, was tested in triplicate and each experiment was
performed at least in triplicate.
Membrane Damage
The level of membrane integrity was studied by staining
with SYTO9 and propidium iodide (PI) according to method
described by O’Neill et al. (2004) with modifications. The
principle of this method is to use two probes staining nucleic
acids: a cell permeable dye (SYTO9) and an impermeant probe
(PI). The permeability of PI is enhanced when cells are dead
or their membrane is impaired. Overnight culture of bacterial
cells in CA-MHB medium was diluted 100 times in fresh
medium and cultured for next 6 h at 37◦C with agitation
(150 rpm). Bacterial cells were centrifuged (2,800 ×g, 10 min),
washed twice and finally diluted in physiological saline (PS) to
0.5 McF (DensiMeter II, EMO, Brno). The inoculum samples
were then mixed with the tested agents (AgNPs, DAP, PR)
to final concentration of 0.5×MBC. Both DAP and PR, two
membrane disrupting agents, were used separately as positive
controls. Bacterial suspension without any antimicrobial agent
added was taken as an untreated control. Inoculum aliquots
(0.1 mL) were transferred immediately into the wells of flat-
bottom black polystyrene 96-well plate (FluoroNuncTM, Thermo
Fisher Scientific). Following incubation at 37◦C (30, 60, and
120 min, without shaking, cultures in wells were combined
with 0.1 mL aliquots of mixed solution of dyes in PS: 60 µM
of PI (Sigma Aldrich) and 10 µM of SYTO9 (Thermo Fisher
Scientific). After stationary incubation (in the dark, RT, 15 min)
fluorescence of green dye (SYTO9) and red dye (PI) was
measured on a microplate reader (EnVision Multilabel Plate
Reader, Perkin Elmer) at Ex/Em = 485/530 and Ex/Em = 485/630,
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respectively. Ratio of green to red fluorescence was calculated
for all samples. The values obtained for samples treated with
agents were compared to those obtained for untreated control
(corresponding to 100% membrane stability in each time point).
To control the number of living bacteria we performed colony
counts for each sample. Each agent was tested in three replicates
and whole experiment was performed at least in triplicate.
Analysis of Structure of Bacterial Cells
With Transmission Electron Microscopy
Transmission electron microscopy (TEM) was used to determine
changes in structure of bacterial cells treated with AgNPs (or
AgNO3), 3ChPL, and their combination. Inocula in late log
phase (cultured for 6 h, at 37◦C and 150 rpm) in CA-MHB
medium (0.5 McF, 1.5 ×108CFU/mL) were treated with agents,
or combinations of agents, at concentration corresponding to
5×MBC for 90 and 180 min (37◦C, 150 rpm). Following
the treatment, the bacterial cells were centrifuged (2,800 ×g,
10 min) and washed twice with phosphate-buffered saline.
Bacterial pellets left in tubes were fixed with 2.5% glutaraldehyde
(Polysciences, Warrington, PA, United States), and then with 1%
osmium tetroxide (Polysciences, Warrington, PA, United States).
After ethanol dehydration, bacteria were embedded in Epon 812
resin (Sigma-Aldrich). The ultramicrotome Leica UC7 was used
to prepare ultrathin sections (55 nm). Lead citrate and uranyl
acetate were added as contrasting agents. The entire study was
performed at 120 kV using Tecnai Spirit BioTWIN microscope
(FEI).
Analyses of Interactions of NQs and
AgNPs
To analyze the interactions between AgNPs and a selected
naphthoquinone (3ChPL), we measured light absorption spectra
in a wide wavelength range of 300–800 nm with 0.5 nm intervals,
using SPECORD 50 Plus Analytik Jena spectrophotometer with
a thermostat (25 ±0.1◦C). Three series of spectrophotometric
titrations were performed: (i) buffer titrated with an increasing
amount of tested compounds (concentration range from 1
to 20 µg/mL for 3ChPL and from 1.8 to 7.4 µg/mL for
AgNPs), (ii) buffer containing 3ChPL (initial concentration
20 µg/mL) titrated with AgNPs (concentration range from 1.8
to 7.4 µg/mL), and (iii) buffer containing tested NQ (initial
concentration 20 µg/mL) titrated with distilled water in a volume
corresponding to the volume of AgNPs solution. Experiments
were done in quartz cuvettes (1 cm light path) containing 2 mL
of 0.2 M sodium-phosphate buffer pH 6.8.
The analyses of the size distribution of aggregates in ddH2O
for AgNPs alone (12.8 µg/mL) and AgNPs with 3ChPL
(12.8 µg/mL with 8 µg/mL, respectively) were performed by
dynamic light scattering (DLS) measurement on Zetasizer Nano
ZS (Malvern, Worcestershire, United Kingdom), by measuring
the intensity of the scattered light. The analyzed solutions were
placed in polystyrene cuvettes. Measurements were conducted
at 25◦C with a He–Ne laser (633 nm, 4 mW), at a 173◦
scattering angle. Results were evaluated using Smoluchowski
approximation, which is known to be rigorously valid only for
spherical-like particles. The obtained data are shown as size
distribution [nm] of light scattering particles (in accordance to
their hydrodynamic diameters) by intensity [%].
Evaluation of Cytotoxicity on Eukaryotic
Cell Line
The human skin keratinocyte cell line HaCaT (CLS order no.
300493) was used to assess the cytotoxicity of tested agents,
and their combinations, by employing the MTT assay, according
to the protocol of Krychowiak et al. (2014). Cell cultures were
treated with a concentration gradient ranging from 1×MBC
to 0.03×MBC of 3ChPL, AgNPs, or AgNO3, as well as with
concentration gradient of 3ChPL combined with AgNPs (0.25×
MBC). The absorbance of formazan was measured at 550 nm
using a microplate reader (Victor 2, 1420 Multilabel Counter,
Perkin Elmer). The cells survival rate was calculated according
to the equation: survival rate (%) = (A – AB/AC– AB)×100,
where A was the absorbance value of treated sample, ABwas
the absorbance value of blank sample (untreated cells, without
formazan salt) and ACwas the absorbance of untreated sample.
Statistical Analysis
The results of biological assays were analyzed for statistical
significance with the Statistica 13 software (StatSoft). The one-
way analysis of variance (ANOVA) followed by the post hoc RIR
Tukey’s test was applied. For pairwise comparisons, a paired
Student’s t-test was performed. Significance level was established
at α= 0.01.
RESULTS
Evaluation of Synergistic Activity of
AgNPs and NQs
The checkerboard Titration Method employed to test the
bactericidal potential of combined NQs and AgNPs revealed
their synergistic interaction in the isobole curves (Figures 2A–C).
Only droserone (MBC = 512 µg/mL) was unable to interact in a
synergistic manner with the AgNPs (FBC index = 1.03; data not
shown). RAM, PL and 3ChPL, all with high anti-staphylococcal
activity (MBC equal to 16, 16, and 8 µg/mL, respectively),
exhibited synergistic bactericidal effect when combined with
AgNPs. For these three NQs the isoboles were placed under the
zero-interaction line and were concave in shape. Moreover, the
FBC indices for all of the compounds were lower than 0.5 (0.28
for PL and RAM, 0.375 for 3ChPL). Noteworthy is the fact that
when the bacterial cells were treated with combined AgNPs and
NQs, the bactericidal effect was observed at significantly lower
concentrations for all of the tested agents. The effective doses of
naphthoquinones were reduced by 75–97%. At the same time,
bactericidal concentration of AgNPs used simultaneously with
NQs decreased by 75–97%. In further experiments we chose the
most active 3-ChPL (MBC = 8 µg/mL) as a representative and
tested the combination of AgNO3and 3ChPL on S. aureus cells.
According to the results shown in Figure 2D, silver ions also
interact with 3ChPL (concave curve, under the zero-interaction
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FIGURE 2 | . Isobole curves depicting synergistic bactericidal effect of silver nanoparticles (AgNPs) and selected naphthoquinones toward Staphylococcus aureus
strain ATCC 25923. (A) Combinatorial effect of 3-chloroplumbagin (3ChPL) and AgNPs; (B) combinatorial effect of plumbagin (PL) and AgNPs; (C) combinatorial
effect of ramentaceone (RAM) and AgNPs; (D) combinatorial effect of 3ChPL and silver nitrate (AgNO3).
line) and the FBC index (0.31) was slightly lower than FBC index
for AgNPs combined with 3ChPL (0.375).
Time-Dependent Killing Efficiency of
Combination AgNPs-NQs
Experiments on time-dependent killing of bacterial cells
confirmed the synergistic interaction of AgNPs and NQs
(Figure 3). First of all, the number of bacterial cells treated
with AgNPs at concentration corresponding to 0.25×
MBC (3.6 µg/mL) combined with 3ChPL at its bactericidal
concentration (8 µg/mL) dropped significantly. Furthermore,
the inoculum size in wells supplemented with combination
of agents was about four logarithms lower after just 4 h in
comparison to samples treated with each agent separately.
Anti-Staphylococcal Potential of
AgNPs-NQs Combination Toward
Antibiotic-Resistant Clinical Isolates
To examine the effect of interaction of AgNPs and 3ChPL on
clinical isolates of S. aureus with different profiles of antibiotic
resistance, experiments were carried out on bacterial cells treated
with the given combination of agents. The results clearly depict
that the synergistic effect is observed also for strains with
resistance to antibiotics (Table 1). For all of the selected isolates
the FBC index was equal to or even lower than 0.375 in spite of
their resistance profile. This result emphasize the potential of the
AgNPs-NQ combination to combat antibiotic-resistant strains of
S. aureus.
In Vitro Cytotoxicity Assessment
The aim of these experiments was to assess the preliminary
therapeutic potential of AgNPs-NQs. The results obtained for
eukaryotic cells treated with different forms of silver clearly
demonstrated that silver nanoparticles were non-toxic toward
eukaryotic cells in the tested range of concentrations (from
0.03×MBC to 1×MBC) whereas silver nitrate was highly
cytotoxic toward the HaCaT cells (Figure 4A). Cell viability
was significantly decreased (24.54 ±1.20%) even when they
were challenged with the lowest tested concentration of AgNO3
(0.5 µg/mL). It is also worth emphasizing that IC50 value of
AgNO3was extremely low (<0.5 µg/mL) and significantly lower
than the IC50 value of 3ChPL (2.2 µg/mL) (Figure 4B).
The viability measured by the MTT assay was relatively
low when the cells were treated with 3ChPL at its minimal
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FIGURE 3 | Changes in time-dependent killing efficiency of naphthoquinone after addition of AgNPs with the example of 3ChPL tested toward S. aureus strain ATCC
25923. Curves were obtained for concentrations of agents used alone or in combinations equal to 1×MBC (8 µg/mL) for 3ChPL and 0.25×MBC (3.6 µg/mL) for
AgNPs. Results are reported as mean values of 9 replicates ±SD. Values indicated with similar letters are significantly different from each other (p<α,α= 0.01).
TABLE 1 | Summary of checkerboard assays for combination of 3-ChPL and AgNPs tested toward the reference strain of S. aureus and clinical isolates resistant to
antibiotics.
S. aureus strains FBC index 3ChPL AgNPs
FBC (µg/mL) Reduction of MBC (%) FBC (µg/mL) Reduction of MBC (%)
ATCC 25923 0.375 1 87.5 3.6 75
703 ko0.375 0.5 87.5 0.9 75
614 k o,c0.375 2 87.5 0.45 75
56/AS c,v0.25 1 87.5 0.45 87.5
6,347o,c,v0.375 0.5 87.5 0.9 75
o, oxacillin-resistant; c, ciprofloxacin-resistant; v, vancomycin-resistant; FBC, fractional bactericidal concentration, bactericidal concentration in combination; MBC, minimal
bactericidal concentration.
FIGURE 4 | Dose-dependent changes of cytotoxicity of the tested agents toward HaCaT cells cultured in vitro.(A) Viability of cells treated with AgNPs and AgNO3
at range of their concentrations from 0.03×MBC to 1×MBC; (B) viability of cells treated with 3ChPL in range of its concentration (0.03×MBC to 1×MBC) both
alone and combined with AgNPs at 0.25×MBC. Minimal bactericidal concentrations (1×MBC) of agents tested separately were equal to 14.4 µg/mL (AgNPs),
8µg/mL (3ChPL), and 16 µg/mL (AgNO3).
bactericidal concentration (MBC = 8 µg/mL) as shown in
Figure 4B. However, the survival rate of HaCaT cells was
significantly higher when concentrations of 3ChPL ranged from
0.25 to 1 µg/mL. A subsequent experiment carried on HaCaT
cells treated with the same concentration range of 3ChPL
but supplemented with AgNPs (0.5×MBC = 7.2 µg/mL),
revealed that the toxicity of the NQ was not enhanced by
AgNPs.
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FIGURE 5 | Time-dependent damage of bacterial cell membrane after
treatment with AgNPs, daptomycin (DAP) and protegrin (PR) at their
concentrations corresponding to 0.5×MBC. Experiments were performed on
S. aureus strain ATCC 25923. Results are reported as mean values of 9
replicates ±SD. Values indicated with similar letters are significantly different
from each other (p<α,α= 0.01).
Damage Detection in Bacterial Cell
Membrane
The procedure of cell staining with SYTO9 and PI was used to
determine the bacterial cell membrane stability after treatment
with AgNPs. Two antimicrobials, DAP and PR, known for their
potential to interact and disrupt bacterial cells membranes, were
used as a control. Each agent was tested at a concentration equal
to 0.5×MBC (AgNPs: 6.2 µg/mL, DAP: 4 µg/mL, PR: 8 µg/mL)
to follow the changes during time course experimentation
(Figure 5). AgNPs led to a slight drop in membrane integrity
during the first 60 min of incubation (membrane integrity level
equal to 72.95 ±4.91%), followed by a statistically significant
disruption after 120 min (membrane integrity level equal to
7.85 ±2.72%). The curve representing the results obtained for
DAP used as a control agent, showed only a slight drop over the
entire time course (to 80.81 ±8.02% after 120 min). The integrity
of bacterial cell membranes treated with PR was significantly
impaired after 30 min and slightly decreased after next 90 min
(to 13.84 ±6.67%). To confirm that decreased ratio of green
to red fluorescence did not correlate with bacterial cells killing,
we prepared cell counts after 120 min treatment. The average
number of bacterial cells after 120 min incubation without and
with AgNPs, DAP and PR was 6.87 ×106, 4.8 ×106, 6.23 ×106,
5.33 ×106CFU/mL, respectively.
Subsequent TEM analysis of bacterial cell structure
confirmed the aforementioned changes in membrane integrity
(Figures 6A,C,D). The surface of S. aureus cells treated with
the AgNPs (alone or in combination with 3ChPL) was covered
with aggregates of nanoparticles and the AgNPs clusters were
also found inside the exosome-like structures made of cell
membrane fragments. Moreover, the way of folding of the
intracellular membrane was observed and most of the bacterial
cells contained mesosome-like structures. Only small changes
(moderate aggregation in the bacterial cytoplasm) appeared
in bacterial cells treated with 3ChPL alone (Figure 6B). When
3ChPL was used simultaneously with AgNPs, the structural
changes were similar to those observed for cells treated with
AgNPs alone. Alterations in the structure of bacterial cells treated
with AgNO3(alone and in combination with 3ChPL) were subtle
(Figures 6E,F). We observed only weak internal aggregation in
the cells. Nanoparticles presented in Figures 6E,F were formed
from AgNO3reduced in the applied culture conditions.
Analysis of Interactions of AgNPs and
NQ
To verify whether the AgNPs and 3ChPL interact (directly and
non-covalently), we analyzed their absorbance spectra alone and
in combination. Since the overlapping spectra of NQ and AgNPs
prevent complete thermodynamic analysis, spectra registered for
3ChPL were subtracted from those registered for 3ChPL titrated
with silver nanoparticles (concentration range: 1.8–9.2 µg/mL).
Significant red shifts in the maxima of absorbance obtained for
AgNPs added to buffer containing 3ChPL, suggest that these
two agents do interact (Figure 7A). Additionally, the analysis
of changes in the hydrodynamic diameter of AgNPs in the
presence of NQ confirmed this hypothesis (Figure 7B). The
average hydrodynamic diameter of nanoparticles increased from
10.96 and 42.63 nm to 13.61 and 119.9 nm, respectively, when
8µg/mL of 3ChPL was added to an aqueous solution of AgNPs.
DISCUSSION
Since the emergence of drug resistance, a continuous
development of new therapeutic approaches for microbial
infections has become essential. Strategies based on synergistic
combinations of antimicrobial agents, as well as drug design
based on nanotechnology and phytomedicine can be effective
in overcoming microbial resistance. The scope of our study was
to explore the synergistic potential of the selected NQs of plant
origin and AgNPs toward S. aureus. As little is known about
the mechanism underlying the synergistic effect of AgNPs-NQs
combination, we also verified the role of the main mechanistic
aspects of the bactericidal activity of AgNPs in the observed
synergy when the nanoparticles are used simultaneously with
naphthoquinone.
In this study we determined the in vitro synergistic anti-
staphylococcal activity of the AgNPs and selected NQs, as well
as verified the potential of this combination toward the clinical
isolates of S. aureus with distinct antibiotic resistance profile.
The bactericidal potential of three out of the four studied NQs
was significantly enhanced by AgNPs. 3-chloroplumbagin, as
the most potent anti-staphylococcal naphthoquinone relative to
PL and RAM, was selected for further testing. A synergistic
bactericidal effect was not observed when AgNPs were combined
with DR. Compared to PL, RAM, and 3ChPL, the chemical
structure of DR is characterized by an additional hydroxyl
group. It has already been demonstrated that a number of
polar groups, like OH- residue, play an important role on both
polarity and biological activity of NQs (Munday et al., 2007;
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Krychowiak et al. Anti-staphylococcal Naphthoquinones and Silver Nanoparticles
FIGURE 6 | Structure of bacterial cells treated with particular agents or their combinations for 180 min. (A) Untreated cells; (B) cells treated with
3ChPL at 5×MBC concentration (40 µg/mL); (C) cells treated with AgNPs at 5×MBC concentration (72 µg/mL); (D) cells treated with combination of 3ChPL and
AgNPs (each at concentration 5×MBC); (E) cells treated with AgNO3at 5×MBC concentration (80 µg/mL); (F) cells treated with combination of 3ChPL and
AgNO3(each at concentration 5×MBC); Experiments were performed for the S. aureus strain ATCC 25923. Black arrows depict structural changes in bacterial
cells: M, mesosome-like structures; E, exosome-like structures.
Camara et al., 2008;Castro et al., 2008). The relationship between
the structure of NQs and the synergistic interaction with the
AgNPs along with in vivo studies on toxicity and anti-infectious
potential will be further investigated. The synergistic bactericidal
effect observed for 3ChPL and silver nitrate indicates that silver
ions can play an important role in the enhancement of NQs’
bactericidal activity. It confirms that the direct toxic effect of
AgNPs on microbial cells depends on silver ions appearing
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Krychowiak et al. Anti-staphylococcal Naphthoquinones and Silver Nanoparticles
FIGURE 7 | Results of analyses of the interactions of AgNPs and
naphthoquinones. (A) The changes of the maximum absorbance points of
spectra of the 3ChPL (initial concentration 20 µg/mL) titrated with AgNPs
(concentration range 1.8–7.4 µg/mL). (B) Changes in hydrodynamic diameter
size of nanoparticles measured by DLS in the presence of 3ChPL. Red line
corresponds to average hydrodynamic diameter size distribution in a
polydispersive population of AgNPs (initial concentration 12.3 µg/mL), peaks
size: 10.96 and 42.63 nm. Green line represents the average hydrodynamic
diameter size distribution of mixture containing AgNPs and 3ChPL (final
concentrations 12.2 and 8 µg/mL, respectively), peaks size: 13.61 and
119.9 nm.
inside the cell and in the cell milieu where metallic silver is
oxidized to silver ions (Xiu et al., 2012). Time-dependent killing
efficiency of 3ChPL was significantly enhanced when 3ChPL
was combined with AgNPs, thus confirming synergistic potential
of the AgNPs-NQ combination. Moreover, the combination of
AgNPs and 3ChPL examined was effective on the clinical isolates
with different resistance profiles. All clinical isolates, resistant
to one or more antibiotics, were susceptible to the combined
treatment.
Moreover, we employed the MTT assay to measure
the cytotoxic effect of the tested antimicrobials and their
combination toward eukaryotic cell cultures. The aim of the
analysis was to preliminarily evaluate the potential role of
AgNPs-NQ as antimicrobial agents. The differences in toxicity
of AgNPs and AgNO3confirmed that silver nanostructures can
be considered as being non-toxic, sources of anti-infectious Ag+
ions that are released from their relatively large surface (Raffi
et al., 2008). What is more, 3ChPL occurred to be less toxic at
and below the value of its fractional bactericidal concentration in
combination with AgNPs. Furthermore, the addition of AgNPs
to the samples treated with 3ChPL did not affect the viability of
cells. These results highlight the potential of the nanoparticles of
silver: not only they improve the antimicrobial activity of NQ
but also help to fine-tune its dose.
To assess the mechanism of the observed synergy
phenomenon, we investigated the role of the disruption of
bacterial cell membrane by AgNPs. The experiments on cells
stained with SYTO9/PI revealed that AgNPs significantly disrupt
S. aureus cell membrane with a rate similar to protegrin-1,
a peptide used as a one of the membrane-disrupting agents
in positive controls. What is more, protegrin-1 combined
with 3ChPL gave an additive bactericidal effect and reduced
bactericidal concentration of this naphthoquinone by 75% (FBC
index = 0.75, data not shown). On the contrary, daptomycin
did not disrupt the membrane significantly and did not
interact with 3ChPL (FBC index = 1.03, data not shown).
This suggests that cell membrane disruption might be one
of the mechanisms that underlie the synergistic effect of
AgNPs and NQs. Since the role of the membrane is to protect
bacterial cell from penetration and adverse effects of toxic
molecules, decrease of membrane integrity would allow
enhanced permeation and likely cell death. For hydrophobic
molecules like naphthoquinones, such mechanism plays an
undeniable role in the enhancement of the antimicrobial
potential (Nikaido, 2003). TEM micrographs obtained in
our study confirmed the changes in bacterial cell membrane
after treatment with AgNPs. Cells treated with nanoparticles
were covered with silver aggregates, formed external vesicles
containing metallic silver and internal mesosome-like structures,
also, they had visible agglomerates in the cytoplasm. Although
bacterial mesosomes are considered to be artificial structures
formed from cell membrane during sample preparation
procedure (Silva et al., 1976), their occurrence had been reported
in bacterial cells treated with agents that impair cytoplasmic
membrane (Shimoda et al., 1995;de León et al., 2010;Rabanal
et al., 2015).
We employed a simple method based on UV-Vis spectroscopy,
followed by DLS measurement to verify whether the AgNPs
interact directly with NQs. Shifts in the absorbance spectra
of NQs titrated with AgNPs along with the extension of
hydrodynamic diameter of two populations of nanoparticles
homoaggregates in the presence of 3ChPL demonstrate
interactions between the studied agents. AgNPs used in this
study were spherical nanostructures stabilized with thioalkane
chains which are responsible for the interaction of nanoparticles
with other chemical compounds (Rana et al., 2012). Furthermore,
it is widely known that the physical interaction (formation of
complexes) of drug and nanoparticles modulates activity and can
play an important role in the final therapeutic effect (Deng et al.,
2016).
Synergistic strategies have been widely studied and
successfully used for many approved pharmaceuticals. In
this paper, we present an approach based on the synergistic
activity of AgNPs and naturally occurring compounds. To the
best of our knowledge, this is the first report exploring enhanced
antimicrobial potential of NQs-AgNPs combination, and also
describes its possible mode of action. The synergistic activity
in this combination has resulted in significant enhancement of
bactericidal activity regardless of antibiotic resistance, together
with a reduction of cytotoxic effect toward the eukaryotic
cells, as well as in a final multi-target antimicrobial effect.
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Krychowiak et al. Anti-staphylococcal Naphthoquinones and Silver Nanoparticles
Through the release of silver ions, interacting with the molecules
of naphthoquinone and disruption of bacterial cell membrane,
the AgNPs enhance the NQs’ anti-staphylococcal activity.
Such complex mechanism of bacterial cells killing reduces the
probability of resistance development (Ulrich-Merzenich et al.,
2009). Synergy testing is an extremely valuable research field
that allows for the design of drug composition active toward
antibiotic-resistant pathogens like S. aureus.
CONCLUSION
In this study, we have demonstrated the potential of AgNPs
to enhance the bactericidal activity of three NQs toward
S. aureus. Moreover, our approach was also effective with
bacterial isolates resistant to many antibiotics. It is a first
report which includes the analysis of the mechanism responsible
for the evaluation of the anti-staphylococcal activity of NQs.
The complexity and efficiency of the combinations examined
could be of value in era of bacterial multi-drug resistance.
The combination of nanotechnology and phytopharmacy is
fast becoming a new research field that is worth exploring
and expanding in order to design new possible treatments for
infectious diseases.
DATA AVAILABILITY
All data supporting the conclusions of this manuscript will be
made available on request.
AUTHOR CONTRIBUTIONS
MK and AKr conceived the original idea, designed the study, and
wrote the manuscript. MK, AKa, MN, and AB performed the
experiments. MK analyzed the data. AB performed the statistical
analysis.
FUNDING
This work was supported by the grant from the National
Science Centre of Poland (PRELUDIUM 10 Grant No.
2015/19/N/NZ7/02802).
ACKNOWLEDGMENTS
Staphylococcus aureus strain ATCC 25923 was obtained from
KPD – the Collection of Plasmids and Microorganisms at the
Department of Biology, University of Gda´
nsk, Poland (strain
designation in KPD collection: KPD 19-BA). The authors would
like to express their gratitude to Michel Perombelon (ex. Scottish
Crop Research Institute, Scotland, United Kingdom) for critically
reviving the manuscript before submission.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fphar.
2018.00816/full#supplementary-material
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2018 Krychowiak, Kawiak, Narajczyk, Borowik and Królicka. This is an
open-access article distributed under the terms of the Creative Commons Attribution
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