ArticlePDF Available

Silver Nanoparticles Combined With Naphthoquinones as an Effective Synergistic Strategy Against Staphylococcus aureus


Abstract and Figures

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.
Content may be subject to copyright.
fphar-09-00816 July 24, 2018 Time: 19:1 # 1
published: 26 July 2018
doi: 10.3389/fphar.2018.00816
Edited by:
Gregory Franklin,
Institute of Plant Genetics (PAN),
Reviewed by:
Xing-Feng Zheng,
Second Military Medical University,
Karthik Siram,
PSG College of Pharmacy, India
Letizia Angiolella,
Sapienza Università di Roma, Italy
Aleksandra Królicka
Specialty section:
This article was submitted to
a section of the journal
Frontiers in Pharmacology
Received: 26 March 2018
Accepted: 09 July 2018
Published: 26 July 2018
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´
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
Frontiers in Pharmacology | 1July 2018 | Volume 9 | Article 816
fphar-09-00816 July 24, 2018 Time: 19:1 # 2
Krychowiak et al. Anti-staphylococcal Naphthoquinones and Silver Nanoparticles
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
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.,
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.
Bacterial Strains and Antimicrobial
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´
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.
Frontiers in Pharmacology | 2July 2018 | Volume 9 | Article 816
fphar-09-00816 July 24, 2018 Time: 19:1 # 3
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 (37C,
150 rpm) in Cation-adjusted Mueller-Hinton Broth (CA-MHB)
prepared from colonies on BHI agar plates (24 h, 37C).
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 37C. Wells with no visible growth
of bacteria were plated out on BHI agar plates and incubated
for 24 h at 37C, 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
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 37C and
150 rpm. Bacterial cells grown overnight were diluted 100 times
in fresh medium and cultured for another 4 h (37C, 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 37C 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 37C (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,
Frontiers in Pharmacology | 3July 2018 | Volume 9 | Article 816
fphar-09-00816 July 24, 2018 Time: 19:1 # 4
Krychowiak et al. Anti-staphylococcal Naphthoquinones and Silver Nanoparticles
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 37C 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 (37C, 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
Analyses of Interactions of NQs and
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 ±0.1C). 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 25C 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.
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
Frontiers in Pharmacology | 4July 2018 | Volume 9 | Article 816
fphar-09-00816 July 24, 2018 Time: 19:1 # 5
Krychowiak et al. Anti-staphylococcal Naphthoquinones and Silver Nanoparticles
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
Frontiers in Pharmacology | 5July 2018 | Volume 9 | Article 816
fphar-09-00816 July 24, 2018 Time: 19:1 # 6
Krychowiak et al. Anti-staphylococcal Naphthoquinones and Silver Nanoparticles
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
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
Frontiers in Pharmacology | 6July 2018 | Volume 9 | Article 816
fphar-09-00816 July 24, 2018 Time: 19:1 # 7
Krychowiak et al. Anti-staphylococcal Naphthoquinones and Silver Nanoparticles
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
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
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.
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
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;
Frontiers in Pharmacology | 7July 2018 | Volume 9 | Article 816
fphar-09-00816 July 24, 2018 Time: 19:1 # 8
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
Frontiers in Pharmacology | 8July 2018 | Volume 9 | Article 816
fphar-09-00816 July 24, 2018 Time: 19:1 # 9
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
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.,
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.
Frontiers in Pharmacology | 9July 2018 | Volume 9 | Article 816
fphar-09-00816 July 24, 2018 Time: 19:1 # 10
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.
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.
All data supporting the conclusions of this manuscript will be
made available on request.
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
This work was supported by the grant from the National
Science Centre of Poland (PRELUDIUM 10 Grant No.
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.
The Supplementary Material for this article can be found
online at:
Babich, H., and Stern, A. (1993). In vitro cytotoxicities of 1,4-naphthoquinone
and hydroxylated 1,4-naphthoquinones to replicating cells. J. Appl. Toxicol. 13,
353–358. doi: 10.1002/jat.2550130510
Barenbaum, M. C. (1978). A method for testing for synergy with any
number of agents. J. Infect. Dis. 137, 122–130. doi: 10.1093/infdis/
Camara, C. A., Silva, T. M., da-Silva, T. G., Martins, R. M., Barbosa, T. P., Pinto,
A. C., et al. (2008). Molluscicidal activity of 2-hydroxy-[1,4]naphthoquinone
and derivatives. An. Acad. Bras. Cienc. 80, 329–334. doi: 10.1590/S0001-
Castro, F. A., Mariani, D., Panek, A. D., Eleutherio, E. C., and Pereira, M. D. (2008).
Cytotoxicity mechanism of two naphthoquinones (menadione and plumbagin)
in Saccharomyces cerevisiae.PLoS One 3:e3999. doi: 10.1371/journal.pone.
CLSI (1996). Methods for Determining Bactericidal Activity of Antimicrobial
Agents; Approved Guideline. CLSI document M26-A. Wayne, PA: Clinical and
Laboratory Standards Institute.
CLSI (2012). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
That Grow Aerobically; CLSI Document M07-A9. Approved Standard, 9th Edn.
Wayne, PA: Clinical and Laboratory Standards Institute.
Dakal, T. C., Kumar, A., Majumdar, R. S., and Yadav, V. (2016). Mechanistic
basis of antimicrobial actions of silver nanoparticles. Front. Microbiol. 7:1831.
doi: 10.3389/fmicb.2016.01831
de León, L., López, M. R., and Moujir, L. (2010). Antibacterial properties
of zeylasterone, a triterpenoid isolated from Maytenus blepharodes, against
Staphylococcus aureus.Microbiol. Res. 165, 617–626. doi: 10.1016/j.micres.2009.
Deng, H., McShan, D., Zhang, Y., Sinha, S. S., Arslan, Z., Ray, P. C., et al. (2016).
Mechanistic study of the synergistic antibacterial activity of combined silver
nanoparticles and common antibiotics. Environ. Sci. Technol. 50, 8840–8848.
doi: 10.1021/acs.est.6b00998
Gaascht, F., Dicato, M., and Diederich, M. (2013). Venus Flytrap (Dionaea
muscipula Solander ex Ellis) contains powerful compounds that prevent and
cure cancer. Front. Oncol. 3:202. doi: 10.3389/fonc.2013.00202
Jain, J., Arora, S., Rajwade, J. M., Omray, P., Khandelwal, S., and Paknikar, K. M.
(2009). Silver nanoparticles in therapeutics: development of an antimicrobial
gel formulation for topical use. Mol. Pharm. 6, 1388–1401. doi: 10.1021/
Juan, L., Zhimin, Z., Lei, L., and Jingchao, Z. (2010). Deposition of silver
nanoparticles on titanium surface for antibacterial effect. Int. J. Nanomedicine
5, 261–267. doi: 10.2147/IJN.S8810
Juniper, B. E., Robins, R. J., and Joel, D. M. (1989). The Carnivorous Plants. London:
Academic Press.
Kreher, B., Neszmelyi, A., and Wagner, H. (1990). Naphthoquinones from Dionaea
muscipula.Phytochemistry 29, 605–606. doi: 10.1016/0031-9422(90)85125-Y
Krolicka, A., Szpitter, A., Gilgenast, E., Romanik, G., Kaminski, M., and
Lojkowska, E. (2008). Stimulation of antibacterial naphthoquinones and
flavonoids accumulation in carnivorous plants grown in vitro by addition of
elicitors. Enzyme Microb. Technol. 42, 216–221. doi: 10.1016/j.enzmictec.2007.
Krolicka, A., Szpitter, A., Maciag, M., Biskup, E., Gilgenast, E., Wegrzyn, G., et al.
(2009). Antibacterial and antioxidant activity of the secondary metabolites
from in vitro cultures of the Alice sundew (Drosera aliciae). Biotechnol. Appl.
Biochem. 53, 175–184. doi: 10.1042/BA20080088
Krychowiak, M., Grinholc, M., Banasiuk, R., Krauze-Baranowska, M., Glod, D.,
Kawiak, A., et al. (2014). combination of silver nanoparticles and Drosera
Frontiers in Pharmacology | 10 July 2018 | Volume 9 | Article 816
fphar-09-00816 July 24, 2018 Time: 19:1 # 11
Krychowiak et al. Anti-staphylococcal Naphthoquinones and Silver Nanoparticles
binata extract as a possible alternative for antibiotic treatment of burn wound
infections caused by resistant Staphylococcus aureus.PLoS One 9:e115727. doi:
Magiorakos, A. P., Srinivasan, A., Carey, R. B., Carmeli, Y., Falagas, M. E.,
Giske, C. G., et al. (2012). Multidrug-resistant, extensively drug-resistant
and pandrug-resistant bacteria: an international expert proposal for interim
standard definitions for acquired resistance. Clin. Microbiol. Infect. 18, 268–281.
doi: 10.1111/j.1469-0691.2011.03570.x
Moreira, C. S., Silva, A. C., Novais, J. S., Sa Figueiredo, A. M., Ferreira, V. F., da
Rocha, D. R., et al. (2017). Searching for a potential antibacterial lead structure
against bacterial biofilms among new naphthoquinone compounds. J. Appl.
Microbiol. 122, 651–662. doi: 10.1111/jam.13369
Munday, R., Smith, B. L., and Munday, C. M. (2007). Structure-activity
relationships in the haemolytic activity and nephrotoxicity of derivatives of
1,2- and 1,4-naphthoquinone. J. Appl. Toxicol. 27, 262–269. doi: 10.1002/
Nikaido, H. (2003). Molecular basis of bacterial outer membrane permeability
revisited. Microbiol. Mol. Biol. Rev. 67, 593–656. doi: 10.1128/MMBR.67.4.593-
O’Neill, A. J., Miller, K., Oliva, B., and Chopra, I. (2004). Comparison of
assays for detection of agents causing membrane damage in Staphylococcus
aureus.J. Antimicrob. Chemother. 54, 1127–1129. doi: 10.1093/jac/
Peterson, L. R. (2009). Bad bugs, no drugs: no ESCAPE revisited. Clin. Infect. Dis.
49, 992–993. doi: 10.1086/605539
Phoenix, D. A., Harris, F., and Dennison, S. R. (2014). Novel Antimicrobial Agents
and Strategies. Weinheim: Wiley-VCH Verlag Gmbh & Co. doi: 10.1002/
Rabanal, F., Grau-Campistany, A., Vila-Farrés, X., Gonzalez-Linares, J., Borràs, M.,
Vila, J., et al. (2015). A bioinspired peptide scaffold with high antibiotic
activity and low in vivo toxicity. Sci. Rep. 5:10558. doi: 10.1038/srep
Raffi, M., Hussain, F., Bhatti, T. M., Akhter, J. I., Hameed, A., and Hasan, M. M.
(2008). Antibacterial characterization of silver nanoparticles againts E. coli
ATCC-15224. J. Mater. Sci. Technol. 24, 192–196.
Rai, M., Yadav, A., and Gade, A. (2009). Silver nanoparticles as a new generation
of antimicrobials. Biotechnol. Adv. 27, 76–83. doi: 10.1016/j.biotechadv.2008.
Rana, S., Bajaj, A., Mout, R., and Rotello, V. M. (2012). Monolayer coated gold
nanoparticles for delivery applications. Adv. Drug Deliv. Rev. 64, 200–216.
doi: 10.1016/j.addr.2011.08.006
Riffel, A., Medina, L. F., Stefani, V., Santos, R. C., Bizani, D., and Brandelli, A.
(2002). In vitro antimicrobial activity of a new series of 1,4-naphthoquinones.
Braz. J. Med. Biol. Res. 35, 811–818. doi: 10.1590/S0100-879X2002000700008
Roe, D., Karandikar, B., Bonn-Savage, N., Gibbins, B., and Roullet, J. B.
(2008). Antimicrobial surface functionalization of plastic catheters by silver
nanoparticles. J. Antimicrob. Chemother. 61, 869–876. doi: 10.1093/jac/dkn034
Shimoda, M., Ohki, K., Shimamoto, Y., and Kohashi, O. (1995). Morphology of
defensin-treated Staphylococcus aureus.Infect. Immun. 63, 2886–2891.
Silva, M. T., Sousa, J. C., Polónia, J. J., Macedo, M. A., and Parente, A. M. (1976).
Bacterial mesosomes. Real structures or artifacts?. Biochim. Biophys. Acta 443,
92–105. doi: 10.1016/0005-2736(76)90493- 4
Thornsberry, C. (1991). Antimicrobial susceptibility testing of anaerobic bacteria:
review, comments, and opinions. Ann. Otol. Rhinol. Laryngol. Suppl. 154, 7–10.
doi: 10.1177/00034894911000S904
Tian, J., Wong, K. K., Ho, C. M., Lok, C. N., Yu, W. Y., Che, C. M., et al.
(2007). Topical delivery of silver nanoparticles promotes wound healing.
ChemMedChem 2, 129–136. doi: 10.1002/cmdc.200600171
Tong, S. Y., Davis, J. S., Eichenberger, E., Holland, T. L., and Fowler, V. G. (2015).
Staphylococcus aureus infections: epidemiology, pathophysiology, clinical
manifestations, and management. Clin. Microbiol. Rev. 28, 603–661. doi: 10.
Ulrich-Merzenich, G., Panek, D., Zeitler, H., Wagner, H., and Vetter, H.
(2009). New perspectives for synergy research with the “omic”-technologies.
Phytomedicine 16, 495–508. doi: 10.1016/j.phymed.2009.04.001
Ventola, C. L. (2015). The antibiotic resistance crisis: part 2: management strategies
and new agents. P T 40, 344–352.
Xiu, Z. M., Zhang, Q. B., Puppala, H. L., Colvin, V. L., and Alvarez, P. J. (2012).
Negligible particle-specific antibacterial activity of silver nanoparticles. Nano
Lett. 12, 4271–4275. doi: 10.1021/nl301934w
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
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms.
Frontiers in Pharmacology | 11 July 2018 | Volume 9 | Article 816
... This suggests that the phyto-fabricated AgNPs may suppress the chemical components in antibiotic discs. The combined activity of the AgNPs and antibiotics was in agreement with previous results for AgNPs using biogenic extracts [45][46][47]. Of note, antibacterial action for the antibiotic-associated AgNPs could be efficient for treating antibiotic-resistant bacteria in humans [21]. ...
... Of note, antibacterial action for the antibiotic-associated AgNPs could be efficient for treating antibiotic-resistant bacteria in humans [21]. [45][46][47]. Of note, antibacterial action for the antibiotic-associated AgNPs could be efficient for treating antibiotic-resistant bacteria in humans [21]. ...
Full-text available
The application of biological materials in synthesizing nanoparticles has become significant issue in nanotechnology. This research was designed to assess biogenic silver nanoparticles (AgNPs) fabricated using two aqueous extracts of Acacia arabica (Arabic Gum) (A-AgNPs) and Opophytum forsskalii (Samh) seed (O-AgNPs), which were used as reducing and capping agents in the NPs development, respectively. The current study is considered as the first report for AgNP preparation using Opophytum forsskalii extract. The dynamic light scattering, transmission electron microscopy, and scanning electron microscopy were employed to analyze the size and morphology of the biogenic AgNPs. Fourier transform infrared (FTIR) spectroscopy and chromatography/mass spectrometry (GC-MS) techniques were used to identify the possible phyto-components of plant extracts. The phyto-fabricated NPs were assessed for their antibacterial activity and also when combined with some antibiotics against Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa and Escherichia coli (Gram-negative) and their anticandidal ability against Candida albicans using an agar well diffusion test. Furthermore, cytotoxicity against LoVo cancer cell lines was studied. The results demonstrated the capability of the investigated plant extracts to change Ag+ ions into spherical AgNPs with average size diameters of 91 nm for the prepared O-AgNPs and 75 nm for A-AgNPs. The phyto-fabricated AgNPs presented substantial antimicrobial capabilities with a zone diameter in the range of 10–29.3 mm. Synergistic effects against all tested strains were observed when the antibiotic and phyto-fabricated AgNPs were combined and assessed. The IC50 of the fabricated O-AgNPs against LoVo cancer cell lines was 28.32 μg/mL. Ten and four chemical components were identified in Acacia arabica (Arabic Gum) and Opophytum forsskalii seed extracts, respectively, by GC-MS that are expected as NPs reducing and capping agents. Current results could lead to options for further research, such as investigating the internal mechanism of AgNPs in bacteria, Candida spp., and LoVo cancer cell lines as well as identifying specific molecules with a substantial impact as metal-reducing agents and biological activities.
... Bacterial cell membrane disruption is supposedly the mechanism underlying the synergistic effects of 3ChPL and AgNPs. Synergistic interactions with AgNPs increased the efficacy of reduced 3ChPL concentrations, at which they are non-toxic to human keratinocytes (Krychowiak et al., 2018). Further studies have shown that AgNPs overcome intrinsic microbial resistance to NQs (Krychowiak-Maśnicka et al., 2021). ...
Full-text available
Plant cell and organ cultures are potential sources of valuable secondary metabolites that can be used as food additives, nutraceuticals, cosmeceuticals, and pharmaceuticals. Phytochemical biosynthesis in various in vitro plant cultures, in contrast to that in planta, is independent of environmental conditions and free from quality fluctuations. Pharmaceutical application of plant biotechnology is of interest to almost all departments of the Faculty of Pharmacy and Institute of Pharmacology in Poland with a botanical profile (Pharmaceutical Botany, Pharmacognosy, and Pharmacology). This study discusses the advances in plant biotechnology for the production of known metabolites and/or biosynthesis of novel compounds in plant cell and organ in vitro cultures in several scientific centers in Poland.
... Several studies have depicted the effective application of silver nanoparticles in conjugation with phytochemicals. Conjugation of silver nanoparticles with 3-chloroplumbagin showed a high synergistic effect with fractional bactericidal concentration index of 0.375 against the S. aureus strains (Krychowiak et al., 2018). Similarly, silver nanoparticles synthesized by using the aqueous extract of Swertia paniculata were more effective than the conventional application of the medicinal herbs against Pseudomonas aeruginosa and Klebsiella pneumoneae (Ahluwalia et al., 2018). ...
Nowadays, the pharma and food industries have been gearing up to meet the urgent need for anti-infective and anti-inflammatory nutritional formulations. In this way, several nutraceutical compounds are being re–evaluated due to their established bioactivities. Few compounds have been or may be efficiently targeted against infections, inflammatory conditions and for immune modulation. However, for successful management of these metabolic conditions, the nutraceuticals need to be designed into effective nutritional formulations. Over recent years there have been tremendous progress in the re-engineering of structurally delivery vehicles which provide stability, enhance bioaccessibility and bioavailability of these compounds. In this perspective, this review focuses on the structural and functional aspects of several such bio-based delivery vehicles like the micro and nano particles, nano-emulsions and liposome-based models. The aim is to bring forth recent information on the efficacious nutraceuticals and the suitable delivery vehicles which would be useful against infections and inflammatory conditions.
... INPs inhibit bacterial growth through different mechanisms [91,92]. However, combinatorial treatments of INPs with antibiotics, polymers, and antimicrobial peptides, can produce a synergistic effect and reduce the therapeutic doses [93,94]. ...
Full-text available
Since the discovery of antibiotics, humanity has been able to cope with the battle against bacterial infections. However, the inappropriate use of antibiotics, the lack of innovation in therapeutic agents, and other factors have allowed the emergence of new bacterial strains resistant to multiple antibiotic treatments, causing a crisis in the health sector. Furthermore, the World Health Organization has listed a series of pathogens (ESKAPE group) that have acquired new and varied resistance to different antibiotics families. Therefore, the scientific community has prioritized designing and developing novel treatments to combat these ESKAPE pathogens and other emergent multidrug-resistant bacteria. One of the solutions is the use of combinatorial therapies. Combinatorial therapies seek to enhance the effects of individual treatments at lower doses, bringing the advantage of being, in most cases, much less harmful to patients. Among the new developments in combinatorial therapies, nanomaterials have gained significant interest. Some of the most promising nanotherapeutics include polymers, inorganic nanoparticles, and antimicrobial peptides due to their bactericidal and nanocarrier properties. Therefore, this review focuses on discussing the state-of-the-art of the most significant advances and concludes with a perspective on the future developments of nanotherapeutic combinatorial treatments that target bacterial infections.
... These compounds show synergy with AgNPs and have an increased cytotoxic effect against Staphylococcus aureus (Li et al., 2011). Combination agents of AgNPs and naphthoquinones suggest a potential strategy to control antibiotic-resistant bacteria such as S.aureus (Krychowiak et al., 2018). ...
Plant derived drugs or formulations have always been explored because of their lower side effects and toxicities compared to synthetic drugs and they have been widely used as traditional and complementary medicines for the management of many diseases including cancer. The major challenges faced were the absorption of the plant-derived drugs, their stability, bioavailability, and transport to the intended sites inside the body. Recent progress in nanotechnology has helped to minimize these limitations and hence phyto-nanoformulations are slowly growing in preclinical trials as well as clinical use. The use of various nanostructures such as nano-micelles, lipid nanoparticles, carbon nanotubes, polymer nanoparticles, and nanoliposomes and various types of drug delivery vehicles such as polybutylcyanoacrylate, polylactic-co-glycolic acid, and lactoferrin has immensely helped in increasing the effectiveness of phytochemical drugs by increasing their stability, better pharmacokinetics and reducing the toxicity and side effects. Phyto-nanoformulations having natural product components such as curcumin, piperine, quercetin, berberine, scutellarin, baicalin, stevioside, silybin, gymnemic acid, naringenin, capsicum oleoresin, emodin, and resveratrol have been shown to improve the condition of patients diagnosed with diseases such as neurodegenerative disorders, diabetes, infection, and cancer. Phyto nanoformulations can also be used to treat disorders of the brain where the blood-brain barrier is impervious to the drugs. These phyto- nanoformulations have been shown to target several molecular cell-signaling and metabolic pathways. This chapter covers the compositions of phyto-nanoformulations and how they have been used to control several diseases.
... Both AMPs and AgNPs have demonstrated their potential in combating resistant microbes (Lara et al., 2010;Rai et al., 2012;Roque-Borda et al., 2021), and both have also been found to act synergistically with conventional antibiotics (Cassone and Otvos, 2010;Wan et al., 2016;Krychowiak et al., 2018;Ruden et al., 2019;Zharkova et al., 2019;Duong et al., 2021). Moreover, it has previously been reported by us as well as by others (Ruden et al., 2009;Salouti et al., 2016), that AMPs and AgNPs can enhance each other's antimicrobial activity, when used in combination. ...
Full-text available
Silver nanoparticles (AgNPs) and antimicrobial peptides or proteins (AMPs/APs) are both considered as promising platforms for the development of novel therapeutic agents effective against the growing number of drug-resistant pathogens. The observed synergy of their antibacterial activity suggested the prospect of introducing antimicrobial peptides or small antimicrobial proteins into the gelatinized coating of AgNPs. Conjugates with protegrin-1, indolicidin, protamine, histones, and lysozyme were comparatively tested for their antibacterial properties and compared with unconjugated nanoparticles and antimicrobial polypeptides alone. Their toxic effects were similarly tested against both normal eukaryotic cells (human erythrocytes, peripheral blood mononuclear cells, neutrophils, and dermal fibroblasts) and tumor cells (human erythromyeloid leukemia K562 and human histiocytic lymphoma U937 cell lines). The AMPs/APs retained their ability to enhance the antibacterial activity of AgNPs against both Gram-positive and Gram-negative bacteria, including drug-resistant strains, when conjugated to the AgNP surface. The small, membranolytic protegrin-1 was the most efficient, suggesting that a short, rigid structure is not a limiting factor despite the constraints imposed by binding to the nanoparticle. Some of the conjugated AMPs/APs clearly affected the ability of nanoparticle to permeabilize the outer membrane of Escherichia coli , but none of the conjugated AgNPs acquired the capacity to permeabilize its cytoplasmic membrane, regardless of the membranolytic potency of the bound polypeptide. Low hemolytic activity was also found for all AgNP-AMP/AP conjugates, regardless of the hemolytic activity of the free polypeptides, making conjugation a promising strategy not only to enhance their antimicrobial potential but also to effectively reduce the toxicity of membranolytic AMPs. The observation that metabolic processes and O 2 consumption in bacteria were efficiently inhibited by all forms of AgNPs is the most likely explanation for their rapid and bactericidal action. AMP-dependent properties in the activity pattern of various conjugates toward eukaryotic cells suggest that immunomodulatory, wound-healing, and other effects of the polypeptides are at least partially transferred to the nanoparticles, so that functionalization of AgNPs may have effects beyond just modulation of direct antibacterial activity. In addition, some conjugated nanoparticles are selectively toxic to tumor cells. However, caution is required as not all modulatory effects are necessarily beneficial to normal host cells.
Raw carrot is known to have antimicrobial activity against Listeria monocytogenes, but the mechanism of action has not been fully elucidated. In this study, we examined carrot antilisterial activity against several strains of Listeria species (including L. grayi, L. innocua, L. seeligeri, and L. welshimeri) and L. monocytogenes. A representative strain of L. monocytogenes was subsequently used for further characterizing carrot antilisterial activity. Exposure to fresh-cut carrot for 15 min resulted in a similar loss of cultivability, ranging from 2.5 to 4.7 log units, across all Listeria strains evaluated. L. monocytogenes recovered from the fresh-cut surface of different raw carrots was 1.6 to 4.1 log lower than levels obtained from paired boiled carrot samples with abolished antilisterial activity. L. monocytogenes levels recovered from fresh-cut carrot were 2.8 to 3.1 log lower when enumerated by culture-dependent methods than by the culture-independent method of PMAxx-qPCR, a qPCR assay that is performed using DNA pre-treated to selectively sequester DNA from cells with injured membranes. These results suggested that L. monocytogenes loss of cultivability on fresh-cut carrot was not associated with a loss of L. monocytogenes cell membrane integrity and putative cell viability. Transmission electron microscopy imaging revealed that L. monocytogenes rapidly formed mesosome-like structures upon exposure to carrot fresh-cut surface but not upon exposure to boiled carrot surface, suggesting there may be an association between the formation of these mesosome-like structures and a loss of cultivability in L. monocytogenes. However, further research is necessary to conclude the causality of this association.
Bacterial infection is a major crisis of 21st era and the emergence of multidrug resistant (MDR) pathogens cause significant health problems. We developed, green chemistry-based silver nanoparticles (G-Ag NPs) using Citrus pseudolimon fruit peel extract. G-Ag NPs has a spherical shape in the range of ~ 40 nm with a surface charge of − 31 Mv. This nano-bioagent is an eco-friendly tool to combat menace of MDR. Biochemical tests prove that G-Ag NPs are compatible with human red blood cells and peripheral blood mononuclear cells. There have been many reports on the synthesis of silver nanoparticles, but this study suggests a green technique for making non-cytotoxic, non-hemolytic organometallic silver nanoparticles with a high therapeutic index for possible use in the medical field. On the same line, G-Ag NPs are very effective against Mycobacterium sp. and MDR strains including Escherichia coli, Klebsiella species, Pseudomonas aeruginosa, and Acinetobacter baumannii isolated from patient samples. Based on it, we filed a patent to Indian Patent Office (reference no. 202111048797) which can revolutionize the prevention of biomedical device borne infections in hospital pre/post-operated cases. This work could be further explored in future by in vivo experimentation with mice model to direct its possible clinical utility.
Growing resistance to currently approved antibiotics is posing serious concern worldwide. The multidrug-resistant organisms are a major cause of mortality and morbidity around the globe. The limited options to treat infections caused by resistant organism requires alternative strategies to increase the effectiveness of antibiotic for better clinical outcomes. Recent advances in nanotechnology have enabled the drugs to be used in nanoscale to increase the effectiveness of antibiotics. The use of nanoparticles to treat infectious diseases has a long history in the pharmaceutical market, and the versatility of these particles to incorporate various materials as carriers make it an attractive option to combat the current crisis of emerging antibacterial resistance. Silver, a metal with many medical applications, has inherent antimicrobial properties. Therefore, silver NPs are appearing as one of the best options to be used in combination with antibiotics to increase effectiveness against resistant bacteria. Here, we discuss the applications and mechanisms of silver NPs to treat microbial resistance in light of recent research.
Vulvovaginal candidiasis (VVC) is a commonly occurring yeast infection caused by Candida species in women. Among Candida species, C. albicans is the predominant member that causes vaginal candidiasis followed by Candida glabrata. Biofilm formation by Candida albicans on the vaginal mucosal tissue leads to VVC infection and is one of the factors for a commensal organism to get into virulent form leading to disease. In addition to that, morphological switching from yeast to hyphal form increases the risk of pathogenesis as it aids in tissue invasion. In this study, jacalin, a phytolectin complexed copper sulfide nanoparticles (NPs) have been explored to eradicate the mono and mixed species biofilms formed by fluconazole-resistant C. albicans and C. glabrata isolated from VVC patients. NPs along with standard antifungals like micafungin and amphotericin B have been evaluated to explore interaction behavior and we observed synergistic interactions between them. Microscopic techniques like light microscopy, phase contrast microscopy, scanning electron microscopy, confocal laser scanning microscopy were used to visualize the inhibition of biofilm by NPs and in synergistic combinations with standard antifungals. Real-time PCR analysis was carried out to study the expression pattern of the highly virulent genes which are responsible for yeast to hyphal switch, drug resistance and biofilm formation upon treatment with NPs in combination with standard antifungals. The current study shows that lectin-conjugated NPs with standard antifungals might be a different means to disrupt the mixed species population of Candida spp. that causes VVC. Lay summary: The present study focuses on exploiting the high biding affinity between the cell surface glycans present in Candida cells and the plant lectin, Jacalin. Jacalin serves as a 'Trojan Horse' wherein the lectin-coupled nanoparticles show a high efficacy when compared with the unconjugated nanoparticles. The present approach also improves the anti-biofilm activity of the antifungal drugs against drug-resistant Candida strains.
Full-text available
Multidrug resistance of the pathogenic microorganisms to the antimicrobial drugs has become a major impediment toward successful diagnosis and management of infectious diseases. Recent advancements in nanotechnology-based medicines have opened new horizons for combating multidrug resistance in microorganisms. In particular, the use of silver nanoparticles (AgNPs) as a potent antibacterial agent has received much attention. The most critical physico-chemical parameters that affect the antimicrobial potential of AgNPs include size, shape, surface charge, concentration and colloidal state. AgNPs exhibits their antimicrobial potential through multifaceted mechanisms. AgNPs adhesion to microbial cells, penetration inside the cells, ROS and free radical generation, and modulation of microbial signal transduction pathways have been recognized as the most prominent modes of antimicrobial action. On the other side, AgNPs exposure to human cells induces cytotoxicity, genotoxicity, and inflammatory response in human cells in a cell-type dependent manner. This has raised concerns regarding use of AgNPs in therapeutics and drug delivery. We have summarized the emerging endeavors that address current challenges in relation to safe use of AgNPs in therapeutics and drug delivery platforms. Based on research done so far, we believe that AgNPs can be engineered so as to increase their efficacy, stability, specificity, biosafety and biocompatibility. In this regard, three perspectives research directions have been suggested that include (1) synthesizing AgNPs with controlled physico-chemical properties, (2) examining microbial development of resistance toward AgNPs, and (3) ascertaining the susceptibility of cytoxicity, genotoxicity, and inflammatory response to human cells upon AgNPs exposure.
Full-text available
Combination of silver nanoparticles (AgNPs) and an antibiotic can synergistically inhibit bacterial growth, especially for the use against drug-resistant bacteria like Salmonella typhimurium. However, the mechanism for the synergistic activity is not known. This study chooses four classes of antibiotics, β-lactam (ampicillin and penicillin), quinolone (enoxacin), aminoglycoside (kanamycin and neomycin), and polykeptide (tetracycline) to explore their synergistic mechanism when combined with AgNPs against the multidrug resistant bacterium Salmonella typhimurium DT 104. Enoxacin, kanamycin, neomycin and tetracycline show synergistic growth inhibition against Salmonella when combined with AgNPs, while ampicillin and penicillin do not. UV-Vis and Raman spectroscopy studies reveal that all these four synergistic antibiotics can form complexes with AgNPs, while ampicillin and penicillin do not. Presence of tetracycline enhances the binding of Ag to Salmonella by 9% and Ag(+) release by 26% in comparison to that without tetracycline, while the presence of penicillin does not enhance the binding of Ag or Ag(+) release. This means that AgNPs first form a complex with tetracycline. The tetracycline-AgNPs complex interacts more strongly with the Salmonella cells and causes more Ag(+) release, thus creating a temporal high concentration of Ag(+) near the bacteria cell wall that leads to growth inhibition of the bacteria. These findings agree with the recent findings that Ag(+) release from AgNPs is the agent causing toxicity.
Full-text available
Staphylococcus aureus is a major human pathogen that causes a wide range of clinical infections. It is a leading cause of bacteremia and infective endocarditis as well as osteoarticular, skin and soft tissue, pleuropulmonary, and device-related infections. This review comprehensively covers the epidemiology, pathophysiology, clinical manifestations, and management of each of these clinical entities. The past 2 decades have witnessed two clear shifts in the epidemiology of S. aureus infections: first, a growing number of health care-associated infections, particularly seen in infective endocarditis and prosthetic device infections, and second, an epidemic of community-associated skin and soft tissue infections driven by strains with certain virulence factors and resistance to β-lactam antibiotics. In reviewing the literature to support management strategies for these clinical manifestations, we also highlight the paucity of high-quality evidence for many key clinical questions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Full-text available
Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.
The way some plants function as carnivores gives insights into plant form, function, and evolution not otherwise readily available. They exhibit features which are common to many other non-carnivorous plants. The extent to which these features have developed, however, and the combination of different features in small organs is unique. The main sections of the book are: the syndrome and the habitat; attraction and trapping; nutrition and digestion; phytochemical aspects; exploitation and mutualism; evolution. -from Publisher
Aims The aims of this study were to design, synthesize and to evaluate 2‐hydroxy‐3‐phenylsulfanylmethyl‐[1,4]‐naphthoquinones against Gram‐negative and Gram‐positive bacterial strains, including methicillin‐resistant Staphylococcus aureus (MRSA) and its biofilm, to probe for potential lead structures. Methods and Results Thirty‐six new analogues were prepared with good yields using a simple, fast, operational three‐procedure reaction and a thiol addition to an ο‐quinone methide using microwave irradiation. All compounds were tested against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis ATCC 15290, Serratia marcescens ATCC 14756, Klebsiella pneumoniae ATCC 4352, Enterobacter cloacae ATCC 23355, Enterococcus faecalis ATCC 29212, S. aureus ATCC 25923, Staphylococcus simulans ATCC 27851, Staphylococcus epidermidis ATCC 12228 and a hospital strain of MRSA. Their antibacterial activity was determined using the disc diffusion method, revealing the activity of 19 compounds, mainly against Gram‐positive strains. Interestingly, the minimal inhibitory concentration ranges detected for the hit molecules (32–128 μg ml⁻¹) were within Clinical and Laboratory Standards Institute levels. Promisingly, compound 15 affected the MRSA strain, with a reduction of up to 50% in biofilm formation, which is better than vancomycin as biofilm forms a barrier against the antibiotic that avoids its action. Conclusions After probing 36 naphthoquinones for a potential antibacterial lead structure against the bacterial biofilm, we found that compound 15 should be explored further and also should be structurally modified in the near future to test against Gram‐negative strains. Significance and Impact of the Study Since vancomycin is one of the last treatment options currently available, and it is unable to inhibit biofilm, the research of new antimicrobials is urgent. In this context, 2‐hydroxy‐3‐phenylsulfanylmethyl‐[1,4]‐naphthoquinones proved to be a promising lead structure against MRSA and bacterial biofilm.
By integrating knowledge from pharmacology, microbiology, molecular medicine, and engineering, researchers from Europe, the U.S. and Asia cover a broad spectrum of current and potential antimicrobial medications and treatments. The result is a comprehensive survey ranging from small-molecule antibiotics to antimicrobial peptides and their engineered mimetics, from enzymes to nucleic acid therapeutics, from metallic nanoparticles to photo- and sonosensitizers and to phage therapy. In each case, the therapeutic approaches are compared in terms of their mechanisms, likelihood to induce resistance, and their efficiency in a global healthcare context. Unrivaled knowledge for professionals in fundamental research, pharmaceutical development and clinical practice.
The ultrastructural study of membrane organization in gram-positive bacteria related to the OsO4 fixation conditions revealed that large, complex mesosomes are observed only when the bacteria are subjected to an initial fixation with 0.1% OsO4 in the culture broth, as in the prefixation step of the Ryter-Kellenberger procedure. Evidence was obtained suggesting that the large mesosomes are produced by this prefixation. The kinetic study of the membrane morphological alterations occurring during the prefixation of Bacillus cereus with 0.1% OsO4 in the culture broth showed that the amount of mesosome material increases linearly from zero to a maximum observed at 1.7 min of prefixation and that at about this time a maximum is reached for the number of mesosomes per unity of cell area and for the average individual mesosome area. The large mesosomes observed in gram-positives fixed by the complete Ryter-Kellenberger procedure would be the result of the membrane-damaging action of 0.1% OsO4. Such damaging action was deduced from the observation that 0.1% OsO4 quickly lyses protoplasts and induces a quick and extensive leakage of intracellular K+ from B. cereus and Streptococcus faeculis. In support of that interpretation is the observation that in bacteria subjected to several membrane-damaging treatments, mesosome-like structures are seen after three different fixation procedures. In bacteria initially fixed with 1% OsO4, 4% OsO4 or 2.5% glutaraldehyde, no large, complex mesosomes are observed, small and simple invaginations of the cytoplasmic membrane being present. The size of these minute mesosomes is inversely proportional that causes of fixation. Uranyl acetate was found among the studied fixatives the one to the rate the least damage to bacterial membranes. This fixative satisfactorily preserves protoplasts. In bacteria initially fixed with uranyl acetate no mesosomes were found. The results of the present work throw serious doubts on the existence of mesosomes, both large and small, as real structures of bacterial cells. It is proposed that a continuous cytoplasmic membrane without infoldings (mesosomes) would be the real pattern of membrane organization in gram-positives.