Nano silver combined with Naphthoquinones has a synergistic effect against Golden Staph S.aureus
Staphylococcus aureus is a human pathogen that causes many antibiotic-resistant infections, such as burn infections, that are life-threatening. Exploring the possible synergy between different antimicrobials, such as nanoparticles and natural plant products, could provide new weapons to combat antibiotic-resistant pathogens. The objective of this study was to examine the potential of nano silver (AgNPs) to enhance the antibacterial activity of selected naphthoquinones (NQs): plumbagin (PL), ramentaceone (RAM), droserone (DR) and 3-chloroplumbagin (3ChPL).
We also attempt to elucidate the mechanism by which nanosilver enhances the antibacterial activity of NQs. We analyzed the interaction of AgNPs with bacterial membranes and its effect on membrane stability (TEM analysis, staining with SYTO9 and propidium iodide), as well as the aggregation of NQs on the surface of the membranes. nanoparticles (UV-Vis spectroscopy and DLS analysis).
Our results clearly demonstrated the synergistic activity of AgNPs and three of the four NQs tested (FBC index ≤ 0.375). This resulted in an increase in their combined bactericidal efficacy against reference S. Aureus and clinical isolates, which differed in resistance profiles.
The synergistic effect (FBC index = 0.375) due to the combination of 3ChPL with silver nitrate used as a control, highlighted the role of the silver ion form released from the nanoparticles in their bactericidal activity resulting compatible with NQs.
The role of membrane damage and AgNPs-NQ interaction in the observed synergy of silver nanoparticles and NQs was also confirmed. Furthermore, the described approach, which is based on the synergistic interaction between the agents mentioned above allows to reduce their effective dose, thereby significantly reducing the cytotoxic effect of NQ on NQ. with eukaryotic HaCaT cells. Therefore, the present study on the combined use of agents (AgNPs-NQs) suggests its potential use as a possible strategy to combat antibiotic resistance of S. aureus.
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1. Introduction
Efforts are put on to discover new methods of fighting infections that are needed in the era of near-antibiotic resistance that we face ( Ventola, 2015 ). Resistance is observed in an increasing number of bacterial and fungal strains in the environment and in clinical practice. Furthermore, the problem of increasing antibiotic resistance is associated with most antibiotics approved for the treatment of infectious diseases ( Magiorakos et al., 2012 ).
Staphylococcus aureus is a Gram-positive bacterium that belongs to a group of the most troublesome antibiotic-resistant pathogens, referred to as “ESCAPE” by Peterson (2009) . Most infections caused by S. aureus .are associated with strains resistant to β-lactams (and other antibiotics) and most of them are burn wound infections ( Tong et al. ., 2015 ). The possibility of treatment failure against infections due to antibiotic resistance is a recurring problem. To solve those problems, it is imperative to develop new, alternative approaches.
Nanotechnology is a rapidly evolving field that can be applied to diverse medical problems. Nano silver (AgNPs), a good example of nanomaterials, are widely studied for their antibacterial activity ( Rai et al., 2009 ). Various formulations containing nanosilver have been proposed to date as antibacterial treatments for burn infection ( Jain et al., 2009 ), to improve wound healing ( Tian et al., 2009). et al., 2007 ), to control implantable infections ( Juan et al., 2010 ), or to avoid medical device-associated infections ( Roe et al., 2008).
The mechanism of antimicrobial action of Nano silver AgNPs is complex and depends on both the nanoparticles and silver ions released from their surface, and involves interactions with many cellular components ( Dakal et al. , 2016 ). Many natural compounds of plant origin also have antibacterial activity and are resistant to antibiotic-resistant pathogens ( Phoenix et al., 2014 ). In the previous study, we demonstrated the synergistic activity of Drosera binata extract when combined with AgNPs against S. aureus ( Krychowiak et al., 2014).
Naphthoquinones (NQs), a class of naphthalene backbone secondary metabolites, were found to be the most abundant and active components among all secondary metabolites detected in the extracts studied. . Droserone, 3-chloroplumbagin, plumbagin, and its isomer ramentaceone (Figure 1 ) are the most common naphthoquinones synthesized in the tissues of plants of the family Droseraceae ( Juniper et al., 1989 ; Kreher et al., 1990 ; Gaascht et al., 2013 ; Krychowiak et al., 2014 ). The antibacterial activity of most NQs is mainly associated with Gram-positive bacteria such as S. aureus , as most Gram-negative bacteria are intrinsically resistant to NQs ( Riffel et al., 2002; Krolicka et al., 2002; Krolicka et al. affairs, 2008 , 2009 ; Moreira et al., 2017 ).
Although the direct use of naphthoquinones as antibacterial agents is limited due to their cytotoxicity against eukaryotic cells ( Babich and Stern, 1993). Therefore, we used a method based on a combination of antibacterial agents to verify the possibility of using NQs as antistaphylococcal agents.
Our study aimed to examine Nano silver AgNPs as agents that enhance the bactericidal capacity of NQs and thereby reduce the effective dose of these secondary metabolites. Furthermore, 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 the membrane damage caused by the nanoparticles. and their interactions with naphthoquinone.
2. Materials and methods
2.1.Bacterial strains and antibacterial agents
In this study, we used one strain S. aureus reference (ATTC 25923) and four isolates from patients (Microbiology Laboratory at Provincial Hospital in Gdańsk, Poland), with resistance profiles. Different antibiotics were established according to the CLSI ( CLSI, 2012 ) guidelines for oxacillin, vancomycin and ciprofloxacin (Supplementary Table 1).
The clinical isolates are 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) resistance). All bacterial strains used in this study are stored in the Laboratory of Bioactive Compounds, Department of Biotechnology, IFB UG and MUG Gdańsk, Poland.
Vancomycin, ciprofloxacin, oxacillin and plumbagin (PL) were purchased by Sigma-Aldrich and Daptomycin (DAP) from Selleck Chemicals. Protegrin-1 (PR) was synthesized by Lipopharm Sp. z oo (Poland). Ramentaceone (RAM) was obtained from the University of Pretoria, Republic of South Africa. Droserone (DR) and 3-chloroplumbagin (3ChPL) were synthesized at Gdańsk Technical University by Dr. E. Paluszkiewicz, as described by Krychowiak et al. (2014) .
Nano silver solutions were purchased from Prochimia Surfaces Sp. z oo (Poland). As described by the manufacturer, AgNPs are spherical, water-soluble nanostructures coated with (11-Mercaptoundecyl)- N, N, N -trimethylammonium chloride, characterized by an average size of 5.5 nm and dispersion is 15%. The initial concentration of the Nano silver solution was 6.74 × 10 14 NPs/ml equivalent to 615 μg/ml (maximum SPR, aaa max : 420–424 nm).
2.2.Nano silver and NQ . antibacterial test
To characterize the antibacterial activity of all agents tested in this study, alone and in combination, we determined their minimum bactericidal concentration (MBC), which is the lowest concentration of the agent. which, after 24 h, reduced the number of bacterial cells in the original sample by 99.9. % (3 logarithms).
To determine MBC, we used the Microdilutions Broth Method ( Thornsberry, 1991 ) according to the CLSI ( CLSI, 1996) guideline. To that effect, we prepared double dilutions of the tested agents in 96-well plates, with a final volume of 0.1 mL per well. Tested concentrations ranged from 0.125 to 512 μg/mL.
Bacterial inoculum was prepared from late log cultures (37 °C, 150 rpm) in Cation-Modified Mueller-Hinton Medium (CA-MHB) prepared from colonies on BHI agar (24) h, 37°C). The inoculum was diluted in fresh CA-MHB medium to 0.5 McF (according to McFarland criteria) as measured with a densitometer (DensiMeter II, EMO, Brno), corresponding to ∼1.5 × 10 8 CFU. /mL (colony forming units per mL).
The bacterial suspension was then diluted with the medium to a density of 2.5–5 × 10 6 CFU and 10 μL quantities were allocated to each of the 96-well plates. In addition, dilutions containing untreated bacteria were plated out to check the number of bacterial cells in the wells in each experiment. Then, the 96-well plates were incubated without shaking for 24 h at 37 °C.
Wells without bacterial growth were plated on BHI agar plates and incubated for 24 h at 37 °C, colony counting was performed to determine the lowest bactericidal concentration of each agent, or combination. of the actors. Each concentration or combination of concentrations is tested in triplicate and each test is performed at least in triplicate.
2.3.Synergy test
To examine the interaction mechanism of the tested antibiotic combinations (synergistic, additive or antagonistic), we used the Titration Method checkerboard ( Thornsberry, 1991 ). This method is based on testing the combination of two agents at their concentration gradient obtained by dilution twice. The tested concentration range of each agent was from 0.03 × MBC to 2 × MBC.
Such an approach allows to determine the changes (decrease or increase) in the minimum effective concentrations of the analytes after combining them. The mode of action of the two agents used simultaneously is mathematically expressed as a fractional bacterial concentration index (FBC index), calculated by the formula: FBC index = FBC A /MBC A+ FBC B /MBC B , where MBC A and MBC B are the lowest bactericidal concentrations of A and B tested separately, while FBC A and FBC B are the lowest bactericidal concentrations of agents A and B when they used in combination.
The interaction nature of the two agents is described by the value of the FBC index: less than or equal to 0.5 – synergistic interaction, higher than 0.5 but less than or equal to 1 – additive interaction, high more than 1 – antagonistic interaction. The isobole method ( Barenbaum, 1978) was used to characterize the interaction between silver nanoparticles and NQs, whereby the isobole curve is the line connecting the points with the lowest bactericidal concentration of the combined agents
.
2.4.The bactericidal effect is time dependent
In each step of the time-dependent killing assay, bacterial cells were cultured in CA-MHB medium in CA-MHB medium at 37 °C and 150 rpm. Bacterial cells grown overnight were diluted 100-fold in fresh medium and cultured for an additional 4 h (37 °C, 150 rpm) until they reached the mid-log phase.
The bacterial suspension was then diluted in fresh medium to 0.5 McF as measured with a densitometer (DensiMeter II, EMO, Brno), 0.1 mL of the inoculum was then transferred to the plate wells. 24 wells with 1 mL of the medium containing the tested antimicrobial agents. (8 μg/mL 3ChPL or 3.6 μg/mL AgNP) and their combinations (8 μg/mL 3ChPL in combination with 3.6 μg/mL AgNP).
Wells containing antimicrobial media were considered as controls. Each well was sampled (50 μL) for colony counting at the following time points: 0, 1, 2, 4, 6, , and 24 h, and the plates were incubated as described above. Each concentration of agent, or combination of agents, is tested in triplicate, and each test is performed at least in triplicate.
2.5.Membrane damage
The degree of membrane integrity was investigated by staining with SYTO9 and propidium iodide (PI) according to the method described by O’Neill et al. (2004) with modifications. The principle of this method is to use two nucleic acid staining probes: a cell-permeable dye (SYTO9) and a non-permeable (PI) probe.
The permeability of PIs is enhanced when cells die or their membranes are weakened. Bacterial cell culture overnight in CA-MHB medium was diluted 100-fold in fresh medium and cultured for the next 6 h at 37 °C with stirring (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). Then, the cultures were mixed with the tested substances (AgNPs, DAP, PR) to a final concentration of 0.5 × MBC. Both DAP and PR, two membrane disrupting agents, were used separately as positive controls.
The bacterial suspension without any added antibacterial agent was considered an untreated control. The inoculum (0.1 mL) was transferred immediately into the wells of a flat-bottomed black polystyrene 96-well plate (FluoroNunc TM, Thermo Fisher Scientific).
After incubation at 37 °C (30, 60, and 120 min, without shaking, the in-well cultures were combined with 0.1 mL of mixed dye solution in PS: 60 μM PI (Sigma Aldrich) and 10 μM SYTO9 (Thermo Fisher Scientific) After static incubation (in the dark, RT, 15 min), the 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, respectively.
The ratio of green to red fluorescence was calculated for all samples. per time) .To control the number of live bacteria, we performed a colony count for each sample.
2.6.Analysis of the structure of bacterial cells with transmission electron microscopy
Transmission electron microscopy (TEM) was used to identify changes in the structure of bacterial cells treated with AgNPs (or AgNO 3 ), 3ChPL and their combinations. The late log phase inoculum (cultured for 6 h, at 37 °C and 150 rpm) in CA-MHB medium (0.5 McF, 1.5 × 10 8 CFU/mL) was treated with agents, or combinations of substances, at concentrations of 5 × MBC respectively for 90 and 180 min (37 °C, 150 rpm).
After treatment, bacterial cells were centrifuged (2,800 × g, 10 min) and washed twice with phosphate-buffered saline. The bacterial pellets remaining in the tube were fixed with 2.5% glutaraldehyde (Polysciences, Warrington, PA, United States), and then with 1% osmium tetroxide (Polysciences, Warrington, PA, United States).
After dehydration with ethanol, bacteria were embedded in Epon 812 resin (Sigma-Aldrich). The ultra-thin Leica UC7 is used to prepare ultra-thin sections (55 nm). Lead citrate and uranyl acetate were added as contrast agents. The entire study was performed at 120 kV using a Tecnai Spirit BioTWIN (FEI) microscope.
2.7.Analysis of the interaction of NQs and nano silver
To analyze the interaction between nano silver and a selected naphthoquinone (3ChPL), we measured the light absorption spectrum in a wide wavelength range of 300–800 nm with a distance of 0.5 nm, using a SPECORD spectrometer. 50 Plus Analytik Jena with thermostat (25 ± 0.1 °C).
Three series of spectral titrations were performed: (i) the buffer was titrated with increasing amounts of the test compound (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 1.8 to 7.4 μg/mL), and (iii) buffer containing tested NQ (initial concentration 20 μg/mL) was titrated with distilled water in an amount corresponding to the volume of the AgNPs solution.
The experiment was performed in a quartz cuvette (1 cm light path) containing 2 mL 0.
Analyzes of the size distributions of aggregates in ddH 2 O for Nano silver AgNPs (12.8 μg/mL) and AgNPs with 3ChPL (12.8 μg/mL with 8 μg/mL, respectively) were performed. performed by dynamic light scattering (DLS) measurement on a Zetasizer Nano ZS (Malvern, Worcestershire, UK), by measuring the intensity of the scattered light.
The analyzed solutions were placed in polystyrene cuvettes. Measurements were conducted at 25°C using a He-Ne laser (633 nm, 4 mW), at a scattering angle of 173°. The results are evaluated using the Smoluchowski approximation, which is known to be valid only strictly for spherical seeds.
The obtained data are expressed as the size distribution [nm] of the light scattering particles (in accordance with their hydrodynamic diameters) by intensity [%].
2.8.Evaluation of cytotoxicity on eukaryotic cell lines
The human skin keratinocyte line HaCaT (CLS sequence number 300493) was used to evaluate the cytotoxicity of the agents tested and their combinations, using the MTT assay, according to the protocol. by Krychowiak et al. (2014) .
Cell cultures were treated with a concentration gradient from 1 × MBC to 0.03 × MBC of 3ChPL, AgNPs or AgNO 3 , as well as with a concentration gradient of 3ChPL in combination 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).
Cell survival was calculated using the equation: survival rate (%) = (A – A B / A C – A B ) × 100, where A is the absorbance value of the treated sample. , AB is the absorbance value of the blank (untreated cells, no formazan salts) and A C is the absorbance of the untreated sample.
2.9.Statistical analysis
The results of biological tests were analyzed for statistical significance using Statistica 13 software (StatSoft). One-way analysis of variance (ANOVA) followed by RIR Tukey’s post-course test was applied. For pairwise comparison, the test of the paired Student was performed. The significance level was set at α = 0.01.
3. Result
3.1.Evaluation of the synergistic activity of Nano silver and NQs
The checkerboard titration method used to test the bactericidal ability of the combined NQs and AgNPs showed their synergistic interaction in the isobole curves (Figure 2A–C). Only droserone (MBC = 512 μg/mL) was unable to interact synergistically with AgNPs (FBC index = 1.03; data not shown). RAM, PL and 3ChPL, all with high antistaphylococcal activity (MBCs equal to 16, 16 and 8 μg/mL, respectively), exhibited synergistic bactericidal effects when combined with AgNPs.
For these 3 NQs, the isoboles are located below the zero interaction line and have a concave shape.
Furthermore, the FBC indices for all compounds were lower than 0.5 (0.28 for PL and RAM, 0.375 for 3ChPL). Of note is the fact that when bacterial cells were treated with the combined AgNPs and NQ, a bactericidal effect was observed at significantly lower concentrations for all agents tested.
The effective dose of naphthoquinones was reduced by 75-97%. At the same time, the bactericidal concentration of AgNPs used concurrently with NQs decreased by 75-97%. In subsequent experiments, we selected the most active 3-ChPL (MBC = 8 μg/mL) as representative and tested the combination of AgNO3 and 3ChPL on cells S. aureus.
2D , silver ions also interact with 3ChPL (concave curve, below interaction line 0) and the FBC index (0.31) is slightly lower than the FBC index for the resulting AgNPs compatible with 3ChPL (0.375).
FIGURE 2. Isobole curves depicting the bactericidal synergistic effect of silver nanoparticles (AgNPs) and selected naphthoquinones against the strain Staphylococcus aureus ATCC 25923. (A) Combined effect of 3-chloroplumbagin (3ChPL) ) and AgNPs; (B) Combination effect of plumbagin (PL) and AgNPs; (C) Combination effect of ramentaceone (RAM) and AgNPs; (D) Combination effect of 3ChPL and silver nitrate (AgNO 3 ).
3.2.The time-dependent killing effect of the combined nano silver-NQ
Experiments on time-dependent killing of bacterial cells confirmed the synergistic interaction of nano silver and NQs (Figure 3 ). First of all, the number of bacterial cells treated with AgNP at a concentration corresponding to 0.25 × MBC (3.6 μg/mL) in combination with 3ChPL at a bactericidal concentration (8 μg/mL) was reduced. significantly.
Furthermore, the inoculum size in the wells supplemented with the combinations of agents was approximately four logarithms lower after only 4 h compared with samples treated with each agent separately.
FIGURE 3. Changes in naphthoquinone-killing efficiency dependent on time after addition of AgNPs with the example of 3ChPL tested for strain S. aureus ATCC 25923. Curves obtained for agent concentrations used alone or in combination using 1 × MBC (8 μg/mL) for 3ChPL and 0.25 × MBC (3.6 μg/mL) for AgNP. The results are reported as the mean of 9 replicates ± SD. The values indicated by similar letters differ significantly ( p <α, α = 0.01).
3.3.Antistaphylococcal potential of nano silver-NQs combination against antibiotic resistant clinical isolates
To examine the effect of the interaction of nano silver and 3ChPL on S. aureus clinical isolates with different antibiotic resistance characteristics, experiments were performed on the treated bacterial cells. with a given combination of agents.
The results clearly describe that a synergistic effect was also observed for antibiotic-resistant strains (Table 1 ). For all selected isolates, the FBC index was equal to or even lower than 0.375 regardless of their resistance profile. This result highlights the potential of the Nano silver AgNPs-NQ combination in combating antibiotic-resistant strains of S. aureus
.
TABLE 1. Summary of bactericidal tests for 3-ChPL and AgNP combinations tested against S. aureus reference strains and antibiotic-resistant clinical isolates.
3.4.Evaluation of cytotoxicity in vitro
The aim of these experiments was to evaluate the preliminary therapeutic potential of Nano silver AgNPs-NQs. The results obtained for eukaryotic cells treated with different forms of silver clearly demonstrated that silver nanoparticles were not toxic to eukaryotic cells over the tested concentration range (from 0 to 0). .03 × MBC to 1 × MBC) while silver nitrate was highly cytotoxic to HaCaT cells (Figure 4A ).
Cell viability was significantly reduced (24.54 ± 1.20%) even when they were tested with the lowest tested concentration of AgNO 3 (0.5 μg/mL). It should also be emphasized that the IC 50 value of AgNO 3 is extremely low (<0.5 μg/mL) and significantly lower than the IC 50 value of 3ChPL (2.2 μg/mL) (Figure 4B ).
FIGURE 4. Dose-dependent cytotoxicity changes of the agents tested against cultured in vitro HahaCaT cells. (A) Viability of cells treated with AgNPs and AgNO 3 at their concentration range from 0.03 × MBC to 1 × MBC; (B) Viability of cells treated with 3ChPL over its concentration range (0.03 × MBC to 1 × MBC) both alone and in combination with AgNPs at 0.25 × MBC. The minimum bactericidal concentrations (1 × MBC) of the agents were tested separately by 14.4 μg/mL (AgNPs), 8 μg/mL (3ChPL) and 16 μg/mL (AgNO 3 ).
The viability measured by the MTT assay was relatively low when cells were treated with 3ChPL at the minimal bactericidal concentration (MBC = 8 μg/mL) as shown in Fig. However, HaCaT cell survival was significantly higher when 3ChPL concentrations ranged from 0.25 to 1 μg/mL.
A follow-up experiment was performed on HaCaT cells treated with the same 3ChPL concentration range but supplemented with AgNPs (0.5 × MBC = 7.2 μg/mL), which showed that NQ toxicity was not enhanced by AgNPs.
3.5.Detecting the extent of damage in bacterial cell membranes
Cell staining with SYTO9 and PI was used to determine the stability of bacterial cell membranes after treatment with Nano silver AgNPs. Two antibacterial agents, DAP and PR, known for their ability to interact with and disrupt bacterial cell 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 monitor changes during the trial period. (Figure 5).
FIGURE 5. Time damage of bacterial cell membranes after treatment with AgNPs, daptomycin (DAP) and protegrin (PR) at their respective concentrations of 0.5 × MBC. Experiments were performed on strain S. aureus ATCC 25923. Results are reported as the mean of 9 replicates ± SD. The values indicated by similar letters differ significantly ( p <α, α = 0.01).
Nanosilver resulted in a slight decrease in membrane integrity during the first 60 min of incubation (membrane integrity level was 72.95 ± 4.91%), followed by a statistically significant disruption after 120 min (membrane integrity level is 7.85 ± 2.72%). The curve represents the results obtained for DAP used as a control, showing only a slight decrease over the entire time (to 80.81 ± 8.02% after 120 min).
The integrity of the bacterial cell membranes treated with PR was significantly impaired after 30 min and slightly decreased after the next 90 min (to 13.84 ± 6.67%). To confirm that the reduction in the ratio of green to red fluorescence was not correlated with killing bacterial cells, we prepared cell numbers after 120 min of treatment. The mean bacterial cell counts after 120 min of incubation without nanosilver, DAP and PR were 6.87 × 106 , 4.8 × 10 6 , 6.23 × 10 6 , 5.33 × 10 6 CFU/mL. , corresponding.
Subsequent TEM analysis of the bacterial cell structure confirmed the aforementioned changes in membrane integrity (Fig. The surface of S. aureus cells treated with AgNPs (alone or in combination with 3ChPL) was covered with aggregates of nanoparticles and clusters of AgNPs were also found inside the generated exosome-like structures of cell membrane fragments.
Furthermore, folding of the endoplasmic reticulum has been observed, and most bacterial cells have a mesoderm-like structure. Only small changes (moderately aggregated in the bacterial cytoplasm) appeared in the bacterial cells treated with 3ChPL only (Figure 6B).
When 3ChPL was co-administered with AgNPs, structural changes were similar to those observed for cells treated with AgNPs alone. The changes in the structure of bacterial cells treated with AgNO 3 (alone and in combination with 3ChPL) were very subtle (Figure 6E, F ).
We observed only weak internal aggregation in cells. The nanoparticles are presented in Figure 6E, F formed from reduced AgNO 3 under the applied culture conditions.
FIGURE 6. Structure of bacterial cells treated with specific agents or combinations of them for 180 min. (A) Untreated cells; (B) cells were treated with 3ChPL at a concentration of 5 × MBC (40 μg/mL); (C) cells were treated with AgNPs at a concentration of 5 × MBC (72 μg/mL); (D) cells were treated with a combination of 3ChPL and AgNPs (each at 5 × MBC concentration); (E) cells were treated with AgNO 3 at a concentration of 5 × MBC (80 μg/mL); (F) cells were treated with a combination of 3ChPL and AgNO 3 (each cell at a concentration of 5 × MBC); Experiments were performed on S. aureus strain ATCC 25923. Black arrows depict structural changes in bacterial cells: M, mesosome-like structure; E, exosome-like structure.
3.6.Analysis of the interaction of nano silver and NQ
To verify whether Nano silver AgNP and 3ChPL interact (directly and non-covalently), we analyzed their absorption spectra alone and in combination. Since the overlapping spectrum of NQs and AgNPs prevents complete thermodynamic analysis, the registration spectra for 3ChPL are subtracted from the registration spectrum for 3ChPL titrated with silver nanoparticles (concentration range: 1.8). –9.2 μg/mL).
A significant red shift in peak absorbance was obtained for AgNPs added to the 3ChPL-containing buffer, suggesting that these two agents interact (Figure 7A ). In addition, the analysis of the hydrodynamic diameter change of AgNPs in the presence of NQ confirmed this hypothesis (Figure 7B).
The mean hydrodynamic diameter of the nanoparticles increased from 10.96 and 42.63 nm to 13.61 and 119.9 nm, respectively when 8 μg/mL 3ChPL was added to the aqueous solution of AgNPs.
FIGURE 7. Interaction analysis results of AgNPs and naphthoquinone. (A) Change of peak absorption points of the spectrum of 3ChPL (initial concentration 20 μg/mL) titrated with AgNPs (concentration range 1.8–7.4 μg/mL). (B) The hydrodynamic diameter size changes of the nanoparticles were measured by DLS in the presence of 3ChPL. The red line corresponds to the mean hydrodynamic diameter size distribution in a population of polydisperse AgNPs (initial concentration 12.3 μg/mL), peak sizes: 10.96 and 42.63 nm . The green line represents the mean hydrodynamic diameter size distribution of the mixture containing AgNPs and 3ChPL (final concentrations 12.2 and 8 μg/mL, respectively), peak size: 13.61 and 119.9 nm.
4. Discuss
Since the emergence of drug resistance, the continuous development of new treatments for microbial infections has become essential. Strategies based on synergistic combinations of antimicrobial agents, as well as nanotechnology- and phytomedicine-based drug design, may be effective in overcoming microbial resistance.
The scope of our research is to explore the synthetic potential of plant-derived selective NQs and AgNPs against S. aureus. As little is known about the underlying mechanism of the synergistic effect of AgNPs-NQs combination, we also verified the role of key mechanistic aspects of AgNPs bactericidal activity in the synergy reported. observed when nanoparticles were co-administered with naphthoquinone.
In this study, we determined the in vitro antistaphylococcal synergistic activity of silver nanoparticles and selected NQs, as well as verifying the potential of this combination for clinical isolates of S . aureus with distinct antibiotic resistance profiles.
The bactericidal potential of three of the four studied NQs was significantly enhanced by AgNPs. 3-chloroplumbagin, the most potent antistaphylococcal naphthoquinone compared with PL and RAM, was selected for further testing.
A synergistic bactericidal effect was not observed when AgNPs were combined with DR. Compared with PL, RAM and 3ChPL, the chemical structure of DR is characterized by an additional hydroxyl group. It has been shown that some polar groups, such as residual OH-, play an important role for both the polarity and biological activity of NQs ( Munday et al., 2007 ; Camara et al., 2008 ; Castro). et al., 2008).
The relationship between the structure of NQs and the synergistic interaction with AgNPs together with in vivo studies of toxicity and anti-infection will be further investigated. The synergistic bactericidal effect observed for 3ChPL and silver nitrate suggests that silver ions may play an important role in enhancing the bactericidal activity of NQs.
It confirmed that the direct toxic effects of AgNPs on microbial cells depend on silver ions present inside the cell and in the cellular environment where metallic silver is oxidized to silver ions ( Xiu et al. events, 2012).
The time-dependent killing efficiency of 3ChPL was significantly enhanced when 3ChPL was combined with AgNPs, thus confirming the synergistic potential of AgNPs-NQ combination. Furthermore, the combination of AgNPs and 3ChPL was tested to be effective in clinical isolates with different resistance profiles.
All clinical isolates, resistant to one or more antibiotics, were susceptible to combination therapy.
Furthermore, we used the MTT assay to measure the cytotoxic effects of the tested antibacterial agents and their combinations on eukaryotic cell cultures.
The aim of the analysis was to preliminary evaluate the potential role of AgNPs-NQs as antibacterial agents. The difference in toxicity of AgNPs and AgNO 3 confirms that silver nanostructures can be considered non-toxic, as a source of Ag + anti-infection released from their relatively large surface ( Raffi et al. events, 2008).
Furthermore, 3ChPL appears to be less toxic at and below the value of its fractional bactericidal concentration when combined with nanosilver. Furthermore, the addition of AgNPs to 3ChPL-treated samples did not affect cell viability.
These results highlight the potential of silver nanoparticles: they not only improve the antibacterial activity of NQ but also help to regulate its dosage.
To evaluate the mechanism of the observed synergistic phenomenon, we investigated the role of bacterial membrane disruption by AgNPs. Experiments on cells stained with SYTO9/PI showed that nanosilver significantly disrupts S. aureus cell membranes at a similar rate to protegrin-1, a peptide used as one of the disrupting agents. membranes in the positive controls.
Furthermore, protegrin-1 in combination with 3ChPL produced an additive bactericidal effect and reduced the bactericidal concentration of this naphthoquinone by 75% (FBC index = 0.75, data not shown).
In contrast, daptomycin did not significantly disrupt membranes and did not interact with 3ChPL (FBC index = 1.03, data not shown). This suggests that membrane disruption may be one of the mechanisms underlying the synergistic effects of AgNPs and NQs.
Since the role of the membrane is to protect the bacterial cell from invasion and adverse effects of toxic molecules, deterioration of membrane integrity would allow enhanced permeability and potentially cause toxicity. cell death. For hydrophobic molecules such as naphthoquinones,Nikaido, 2003 ).
The TEM micrograph obtained in our study confirmed the changes in the bacterial cell membrane after nanosilver treatment. The nanoparticle-treated cells were covered with silver aggregates, forming outer vesicles containing metallic silver and internal mesosome-like structures, which also had visible aggregates. found in the cytoplasm.
Although bacterial centrosomes are considered to be artificial structures formed from the cell membrane during sample preparation ( Silva et al., 1976 ), their occurrence has been reported in bacterial cells. bacteria treated with cytoplasmic membrane-depleting agents ( Shimoda et al., 1995 ; de León et al., 2010 ; Rabanal et al., 2015 ).
We used a simple method based on UV-Vis spectroscopy followed by DLS measurement to verify whether the silver nanoparticles interact directly with NQ. The change in the absorption spectra of NQs titrated with nanosilver together with the hydrodynamic diameter expansion of the two homogeneous nanoparticle populations in the presence of 3ChPL demonstrate the interaction between the agents studied. rescue.
The AgNPs used in this study are spherical nanostructures stabilized with thioalkane chains responsible for the interaction of the nanoparticles with other chemical compounds ( Rana et al., 2012 ).
Furthermore, it is widely known that physical interaction (complex formation) of drugs and nanoparticles modulates activity and may play an important role in the final therapeutic effect ( Deng et al. events, 2016 ).
Synergistic strategies have been extensively studied and used successfully for many approved pharmaceuticals. In this paper, we present an approach based on the resonance activity of nanosilver and naturally occurring compounds.
To the best of our knowledge, this is the first report to explore the enhanced antibacterial potential of the NQs-AgNPs combination and also describe its possible mode of action. The synergistic activity in this combination resulted in a significant enhancement of bactericidal activity regardless of antibiotic resistance, together with a reduction in cytotoxic effects on eukaryotic cells, as well as in antibacterial effects. ultimate multi-goal.
Through the release of silver ions, interaction with naphthoquinone molecules, and disruption of bacterial cell membranes, AgNPs enhance the anti-staphylococcal activity of NQs.Ulrich-Merzenich et al., 2009 ). Synergy testing is an extremely valuable area of research that enables the design of drug components that are active against antibiotic-resistant pathogens such as S. aureus .
5. Conclusion
In this study, we demonstrated the potential of nanosilver in enhancing the bactericidal activity of three NQs against S. aureus . Furthermore, our approach is also effective against strains of bacteria that are resistant to multiple antibiotics. This is the first report to include an analysis of the mechanism responsible for assessing the antistaphylococcal activity of NQs. The complexity and effectiveness of the combinations examined may be of value in the age of multidrug-resistant bacteria. The combination of nanotechnology and phytopharmacy is rapidly becoming a new research area worth exploring and expanding in order to design new treatments for infectious diseases.
Nguồn tham khảo: Naphthoquinones as an Effective Synergistic Strategy Against Staphylococcus aureus
Marta Krychowiak1, Anna Kawiak2, Magdalena Narajczyk3, Agnieszka Borowik4 and Aleksandra Królicka1*
1Laboratory of Biologically Active Compounds, Intercollegiate Faculty of Biotechnology, Medical University of Gdańsk, University of Gdańsk, Gdańsk, Poland
2Laboratory of Plant Protection and Biotechnology, Intercollegiate Faculty of Biotechnology UG and MUG, University of Gdańsk, Gdańsk, Poland
3Laboratory of Electron Microscopy, Faculty of Biology, University of Gdańsk, Gdańsk, Poland
4Laboratory of Biophysics, Intercollegiate Faculty of Biotechnology UG and MUG, University of Gdańsk, Gdańsk, Poland