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Article

Synergistic Action between Copper Oxide (CuO) Nanoparticles and Anthraquinone-2-Carboxylic Acid (AQ) against Staphylococcus aureus

by
Prakhar Srivastava
,
Yongjun Kim
,
Hyejin Cho
and
Kwang-sun Kim
*
Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan 46241, Republic of Korea
*
Author to whom correspondence should be addressed.
J. Compos. Sci. 2023, 7(4), 135; https://doi.org/10.3390/jcs7040135
Submission received: 7 February 2023 / Revised: 13 March 2023 / Accepted: 27 March 2023 / Published: 3 April 2023
(This article belongs to the Special Issue Bio-Abio Nanocomposites for Bio-Applications, Volume II)
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Abstract

:
Infections linked to Staphylococcus spp. are difficult to treat with current antibiotic therapy, resulting in increased antibiotic resistance populations. One of the leading strategies to overcome this issue is the novel combination of antibacterial nanoparticles (NPs) and sustainable natural compounds. This study reported the identification of a new synergistic combination of copper oxide (CuO) NPs, a well-known antibacterial agent against Staphylococcus spp., and anthraquinone-2-carboxylic acid (AQ), a plant-derived antimicrobial compound, with a potent and specific fashion in killing Staphylococcus spp. CuO NPs were synthesized using the one-pot coprecipitation method and characterized by using X-ray diffraction and transmission electron microscopy. Further checkerboard analysis showed that CuO NPs and AQ increased the antibacterial activity of individual agents against Staphylococcus aureus among Staphylococcus spp. by four- to eightfold compared to individual materials. Additional mechanistic studies on the synergy revealed that the inhibition of biofilm formation and loss of cytoplasmic volume with cell shrinkage are the major actions of the combination in expressing the phenotype. This study clearly showed that the combination of CuO NPs and AQ would be a novel strategy to eradicate S. aureus infections.

1. Introduction

The most common species detected in biofilm-associated infections, such as chronic wounds and superficial skin infections, is Staphylococcus spp., especially Staphylococcus aureus (S. aureus) [1]. The exopolysaccharide-protected architecture of S. aureus allowed bacteria to congregate, increasing the metabolic activity in biofilm populations and decreasing antibiotic diffusion in the biofilm matrix [2]. The development of novel antibacterial therapies is required due to antibiotic resistance and a lack of last-resort treatments [3]. Combination therapy is routinely used to enhance the empiric coverage offered by two or more antimicrobials with different activity spectra against Gram-positive bacterial infections [4]. Furthermore, it takes advantage of avoiding or preventing resistance during single antimicrobial therapy. Due to the expanded spectrum, enhanced antibacterial activity, and reduced use of antibiotics, combination therapy has become one of the most successful ways to treat bacterial infections [5].
Metallic nanoparticles (NPs) and their oxides have been used in many different biomedical areas, including (a) inhibiting vital enzyme activity, (b) sabotaging metabolic pathways, and (c) aggregating metal ions inside bacteria [6]. Especially, inorganic NPs or oxides have been demonstrated as the most effective agents against several bacterial pathogens [7]. Among metallic NPs, copper oxide (CuO) NPs are rapidly gaining interest as they offer significant benefits in medicinal applications, such as cost-effectiveness and ease of manufacture [8]. In addition, CuO NPs have a good ion release activity compared to other noble metallic NPs, improving their solubility in medical use. According to numerous studies, CuO NPs exhibited broad-spectrum antibacterial activity on Gram-positive and Gram-negative bacteria, such as S. aureus, Bacillus spp., Pseudomonas aeruginosa, Proteus spp., and Escherichia coli [9,10,11]. Despite their broad-spectrum action, their limitations must be overcome before being used as viable antibacterial agents, including increased minimum inhibitory concentration (MIC) values and low selectivity for various bacterial species [12,13]. Therefore, the chemical modification of CuO NPs or the combination of CuO NPs with other compounds activates CuO NPs’ function at a comparable level to commercial antibiotics. Because different synthesis protocols have not improved CuO NPs’ activity [14], identifying nontoxic or widely used antimicrobial compounds showing a synergistic action to CuO NPs will be a future direction to enhance the CuO NPs activity as an effective antibacterial agent against pathogens.
Several studies have reported the synergistic use of metallic NPs and their oxides with natural antimicrobial compounds or commercial antibiotics against bacterial infections. For instance, zinc oxide (ZnO) NPs in combination with commercially available antibiotics, such as ampicillin and ciprofloxacin, have increased the susceptibility pattern of E. coli and S. aureus [15]. Another study on titanium oxide (TiO2) NPs with nalidixic acid and commercially available antibiotics (cephalosporin, β-lactam, and tetracycline) against methicillin-resistant S. aureus (MRSA) showed the potential synergistic activity in decreasing the susceptibility pattern of MRSA [16]. Moreover, the synergistic combination of ZnO NPs with carbapenem antibiotics has also shown potential antibiofilm and antibacterial activities against Pseudomonas-associated infection, highlighting the effectiveness of combination drugs against drug-resistant isolates [17]. Most studies have been conducted on silver oxide or ZnO NPs [18,19,20]. However, few works have been conducted on CuO NPs as a synergistic material [21]. Therefore, it is worthwhile to identify the compounds to be used as synergistic partners to CuO NPs to enhance the use of CuO NPs in a novel antibacterial therapeutic strategy against pathogens.
Natural products have been crucial to the growth of antimicrobial drugs, especially for the past 20 to 25 years [22]. These substances have been shown to affect the pathogenicity islands of bacteria, demonstrating their antibacterial potency against diseases [23]. A wide range of naturally occurring organic compounds in plants are called “plant-derived chemicals” [24]. Many studies have shown that they have beneficial antioxidant, antibacterial, and antifungal properties. An extensive family of naturally occurring substances known as anthraquinones has been demonstrated to possess antibacterial, antibiofilm, anti-inflammatory, and antioxidative activities [25,26,27]. Anthraquinones, such as 1,8-dihydroxyanthraquinone [28], 1-hydroxyanthraquinone [29], alizarin [30], and purpurin [31], have been shown to exhibit antibacterial and antibiofilm activities against major fungal and opportunistic bacterial pathogens. Generally, the above anthraquinones have shown an MIC of >200 µg/mL against MRSA, but emodin has better activity at an MIC of 12.5 µg/mL [32]. Moreover, emodin is known to target extracellular matrix production during early-stage biofilm development on Staphylococcus spp. [33,34]. Recently, anthraquinone-2-carboxylic acid (AQ) has been considered a potential biofilm inhibitor and a contender for treating Staphylococcus-related illnesses [32]. However, the antibacterial activity of AQ and its mechanisms have not been explored in detail, except on pathogens, such as Clostridium spp. [35] and E. coli [36]. Furthermore, the remaining question is how AQ can be used in antibacterial therapy against Staphylococcus.
The current market demands efficient antimicrobials targeting infectious pathogens more quickly and effectively [37]. One possible way to meet this requirement is to use drug combinations that target the same distinct pathogenicity islands of infections by antimicrobials. Like AQ, CuO NPs have been shown to prevent S. aureus biofilm formation [38]. Therefore, this study proposed that the synergistic interaction between CuO NPs and AQ may potentiate anti-S. aureus activity primarily via the biofilm architecture of S. aureus. Unfortunately, no report is available on the synergistic use of AQ and a nanostructured antibacterial agent, CuO NPs, to eradicate biofilm-associated infections by Staphylococcus spp.
This study aimed to characterize the synergistic combination of CuO NPs and AQ for increasing the antibacterial activity of CuO NPs against Staphylococcus spp. To this end, the synergistic potentiality of CuO NPs and AQ was performed using checkerboard analysis, and the potential action mechanisms of the combination treatment were observed by using scanning electron microscopy (SEM), biofilm formation assays, and synergy with commercially available pathway-targeted antibiotics against Staphylococcus spp., especially S. aureus. According to these findings, CuO NPs and AQ demonstrated effective synergistic efficacy by preventing biofilm development and altering cell morphology as a key mechanism. This is the first report of a synergistic combination of CuO NPs and AQ, and the combination will be used to develop an effective combination therapy for S. aureus infections. The novel synergistic combination of CuO NPs and AQ can be further exploited to enhance the antibacterial activity against other opportunistic pathogens.

2. Materials and Methods

2.1. Materials

CuO NPs synthesis was carried out using copper (II) chloride dihydrate (CuCl2·2H2O; ACS reagent > 99%; Sigma-Aldrich, St. Louis, MO, USA) and sodium hydroxide (NaOH; pellets 97%; Junsei, Tokyo, Japan). The organic compound AQ (C15H8O4; >99.0%) was purchased from Tokyo Chemical Industry (Tokyo, Japan). CuO NPs and AQ powder were dissolved in 1% dimethylsulfoxide (DMSO; high-performance liquid chromatography grade; Junsei).

2.2. CuO NPs Synthesis

CuO NPs were prepared using a one-pot coprecipitation process with slight modifications [14]. In a typical procedure, 0.5 M CuCl2.2H2O and 1 M NaOH solution were prepared separately as an aqueous solution in 10 mL distilled water (dH2O). With constant stirring, 1 M NaOH was gradually added to 0.5 M CuCl2·2H2O, and the blue precipitate was produced instantly (CuOH2; pH 10). The solution was kept at room temperature overnight with continuous stirring until the black precipitate (CuO NPs) was formed (Figure 1). The precipitate was washed thrice with ethanol and water (1:1) before being vacuum dried at 27 °C. The powdered CuO NPs was stored at room temperature until further characterization.

2.3. Characterization

2.3.1. X-ray Diffraction (XRD) and Transmission Electron Microscopy (TEM)

The structure and crystallinity of CuO NPs were characterized using XRD at room temperature. Using an X-ray diffractometer (D8 Advance with the DAVINCI design; Bruker, Billerica, MA, USA) fitted with an Ni-filtered Cu Kα radiation source (1.5406 Å), the XRD pattern was captured in a 2θ range of 20° to 80°. TEM was used to assess the microstructure of a typical CuO NPs (Bruker Nano GmbH, Berlin, Germany). The carbon-coated 300-mesh Cu grids were used to produce the TEM samples.

2.3.2. Dynamic Light Scattering (DLS) and Zeta Potential Measurement

The physical parameters of samples, such as the size and stability of NPs, were analyzed using Nanopartica SZ-100 (Horiba Ltd., Kyoto, Japan) [39,40]. The samples after the synthesis were diluted to a final concentration of 200 µg/mL using phosphate-buffered saline (PBS; pH 7; LPS Solution, Daejeon, Republic of Korea), and the temperature was maintained at 25 °C. For stability analysis, the zeta potential was measured using samples at the same concentration above either original or stored for 1 month in PBS at 4 °C after the synthesis.

2.3.3. Preparation of Bacterial Cells

Bacterial cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA; https://www.atcc.org; accessed on 29 March 2023) and the Korean Collection for Type Cultures (KCTC; Jeongeup, Korea; https://kctc.kribb.re.kr/; accessed on 29 March 2023). The isolates were prepared on a Luria-Bertani (LB) agar plate for 18 h at 37 °C. The Sensititre nephelometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to make 0.5 McFarland turbidity bacterial suspensions.

2.3.4. MIC Determination and Checkerboard Analysis

The antibacterial activity of CuO NPs and AQ as MIC values was determined as described previously [41] using serially diluted CuO NPs and AQ solutions (31.25–1000 μg/mL) in 1% DMSO on 96-well plates (SPL, Daejeon, Republic of Korea) and bacterial suspensions in Muller–Hinton broth (MHB; MB Cells, Seoul, Republic of Korea) for 18 h at 37 °C without shaking. The fractional inhibitory concentration index (FICI) value for CuO NPs and AQ was determined as described previously [42,43]. The FICI value was calculated according to the formula: FICI = (ICCuO NPs + AQ/ICCuO NPs) + (ICCuO NPs + AQ/ICAQ). The interaction was considered synergistic when FICI was <0.5, additive when FICI was >0.5 to ≤1, antagonistic when FICI was ≥4, and indifferent for intermediate value.

2.3.5. Resazurin Indicator Assay

Resazurin indicator stain (7-hydroxy-3H-phenoxazin-3-one-10-oxide sodium salt, ≥95%; Sigma-Aldrich) to analyze live cells from NPs aggregates, as described previously [44]. A working concentration (0.01%) was added to each grown cell in 96-well plates, and the plates were further incubated for 3 h at 37 °C with vigorous shaking. The images were taken after bacterial growth using Samsung NX-200 (Samsung, Suwon, Republic of Korea).

2.3.6. Biofilm Formation Assay

Biofilm formation was evaluated using a plate calorimetric assay [45]. The ATCC 25923 strain was grown in LB supplemented with 0.5% glucose for 16 h at 37 °C and diluted to OD600 = 0.6. The culture was further diluted (1:100) in fresh LB broth, dispersed in 96-well flat-bottomed plates, mixed with samples (CuO NPs and AQ at 1/2 MIC, 1/4 MIC, 1/8 MIC, and 1/2 FICI), and incubated for 24 h at 37°C without shaking. The plates were gently washed with PBS, air dried, and subjected to 0.1% crystal violet (CV; Sigma-Aldrich) staining. Finally, the stained plates were resuspended with 30% glacial acetic acid, and the relative biofilm formation was evaluated using OD570/OD600 values. The average ± standard deviation (SD; p < 0.05) were shown (n = 7).

2.3.7. Fluorescence Microscopy

The effects of CuO NPs and AQ on S. aureus biofilm architecture were examined using fluorescence microscopy with acridine orange dye (Sigma-Aldrich) [46]. In brief, overnight grown bacterial cells were diluted at 1:100 in LB broth (supplemented with 0.5% glucose) and mixed with dH2O, CuO NPs, AQ, and CuO NPs/AQ at specific concentrations in a six-well plate and incubated at 37 °C for 24 h under static conditions. The adhered cells were rinsed thrice with PBS and left to air dry. Acridine orange (0.01% working solution in dH2O) was used to stain adhered cells, and the plates were then incubated at 37 °C for 10 min. After dye removal and twice PBS washes, embedded biofilm cells were examined using fluorescence microscopy (OPTIKA-B290, Ponteranica, Italy) at a ×40 objective lens.

2.3.8. Morphological Analysis of S. aureus ATCC 25923 with a Synergistic Combination

Morphology analysis of bacterial cells treated with CuO NPs and AQ at MIC (1000 and 125 μg/mL) and synergistic concentration (250 and 15.6 μg/mL) to overnight cultured cells were incubated for 3 h at 37 °C, 250 rpm. The culture was collected by centrifugation at 12,000 rpm for 3 min and resuspended in 500 μL PBS containing 2% formaldehyde and 1% glutaraldehyde. Cells were fixed by incubating samples for 20 min at room temperature. Further, SEM image analysis was performed as described previously [47] using VEGA3, a versatile tungsten thermionic emission SEM system (TESCAN, Fuveau, France).

2.3.9. Mechanistic Evaluation of CuO NPs and AQ with Commercially Available Antibiotics

The combinational activity of CuO NPs and AQ with available antibiotics was evaluated as described previously [48], Briefly, 5 μL CuO NPs and AQ at 1/2 MIC concentration (i.e., 500 and 62.5 μg/mL, respectively) and 1/2 synergistic value (final concentration, 125 and 7.8 μg/mL for CuO NPs and AQ, respectively) were added to 195 μL MHB inoculated with ATCC 25923 cells in a Sensititre staphylococci antibiotic susceptible plate (EUST2; Thermo Fisher Scientific). MIC was determined using the microbroth dilution method. Resazurin indicator stain was further used to differentiate NPs aggregates and grown cells.

2.3.10. Statistical Analysis

Statistical analyses for the CV assay were performed using Origin 6.1 (OriginLab, Corp., Northampton, MA, USA). Biological replicate data were presented as the average ± SD.

3. Results and Discussion

3.1. Material Properties

3.1.1. XRD Pattern

The crystalline phases of the CuO NPs sample were evaluated using XRD (Figure 2). XRD reflection peaks of the synthesized CuO NPs at 32.45°, 35.50°, 38.7°, 46.27°, 48.70°, 51.2°, 53.48°, 58.24°, 61.51°, 66.17°, 68.04°, 72.36°, and 75.09° corresponded to the (110), (002), (110), (111), (200), (112), (202), (113), (311), (022), (220), (311), and (004) planes, respectively [14]. These peaks were consistent with the crystalline CuO nanostructure (JCPDS card no- 41-0254). Hence, XRD data confirmed CuO NPs production using an easy one-pot coprecipitation approach.

3.1.2. Morphological Analysis

The high-resolution TEM (HR-TEM) image of the CuO NPs sample showed distinct lattice fringes with an interplanar distance of 0.27 nm (Figure 3), corresponding to the (110) plane indicated as ultrathin NPs, as described previously [49]. Hence, HR-TEM results indicated the existence of the nanoflower CuO structure, as validated by using XRD outcomes (Figure 2).

3.1.3. Size and Stability of CuO NPs

The size of synthesized CuO NPs prepared in this study was measured using DLS. From the measurements (Figure 4a), CuO NPs were heterogeneous in size, with 84.8% of the particles in the 837 to 1967 nm range. In addition, the mean size of 1479.2 ± 456.9 nm (~1.4 μm) with maximally frequent samples was 1285 nm, suggesting that CuO NPs synthesized in this study are mainly a two-dimensional nanosheet, as characterized using TEM in a previous study [14]. Further, the stability and charge on the surface of synthesized CuO NPs were assessed by using zeta potential analysis [50], which measures the magnitude of the repulsion or attraction between particles. The surface potential of NPs is responsible for the behavior of colloidal NPs in aqueous solutions due to their ionic characteristics. The mean zeta potential values of CuO NPs either after the original (after the synthesis) or its storage in PBS at 4°C for 1 month were 38.3 ± 0.72 and 36.8 ± 1.31 mV, respectively, which deviated by <4% (Figure 4b), indicating that synthesized CuO NPs are stable cationic materials.

3.2. Antibacterial Activity

3.2.1. Evaluation of Antibacterial Activity

The MIC of CuO NPs and AQ against the S. aureus (ATCC 25923) strain was determined using a 96-well plate broth microdilution method, corroborating with the resazurin viability assay. In Figure 5, the MIC values of AQ and CuO NPs against ATCC 25923 were 125 and 1000 µg/mL, respectively. The efficacy of CuO NPs and AQ to two MRSA strains is shown in Table 1. Results showed that the MIC of AQ against MRSA1 (Figure S1b) and MRSA2 (Figure S1c) was 250 µg/mL (Figure S1b,c), indicating that the antibacterial activity of AQ against MDR strains was less effective than that of the non-MDR S. aureus strain. Next, the antibacterial activity of AQ and CuO NPs against other Staphylococcus spp. was evaluated to see the selectivity of both materials. To this end, Staphylococcus epidermidis (Figure S1d,e) and Staphylococcus saprophyticus (Figure S1f), which reside together with S. aureus as common skin pathogens [51], were selected, and the MIC of both materials was determined. Results showed that CuO NPs against all Staphylococcus strains showed the same activity (MIC = 1000 µg/mL; Table 1).
Meanwhile, AQ against type strains of S. epidermidis (KCTC 13171; Figure S1d) and S. saprophyticus (ATCC 15305; Figure S1f) is the same activity (MIC = 125 µg/mL) as S. aureus, whereas AQ in S. saprophyticus is twofold less active (MIC = 250 µg/mL) than S. aureus and S. epidermidis. The above data indicated that S. aureus and S. epidermidis are better targets for the synergistic action between CuO NPs and AQ. Furthermore, the effects of MDR S. aureus and S. epidermidis were assessed on CuO NPs and AQ. In Table 1, CuO NPs and AQ against MDR S. epidermidis showed twofold more activity (MIC = 62.5 µg/mL) than type strain, whereas AQ was less active to MDR S. aureus. These findings suggested that AQ shows a preference for MDR S. epidermidis, a commensal pathogen that does not produce enterotoxins, unlike S. aureus [52]. Comparatively, the clinical type of S. epidermidis (Figure S1e) is less virulent; therefore, it is less prominent in antibiotic resistance and easily targeted by potential antimicrobial agents [53].

3.2.2. Checkerboard Analysis of CuO NPs/AQ Combination against S. aureus

In Table 1, S. aureus (MRSA1 and MRSA2), S. epidermidis (ATCC 12228), and S. saprophyticus ATCC 15305 were also tested for the synergistic action of CuO NPs with AQ by checkerboard analysis. However, the FICI value was >0.5 (Table 2). Of all these isolates, S. aureus ATCC 25923 showed good synergy based on FICI values and was taken further for the subsequent work. Figure 6 highlights the four- or eightfold increased antibacterial activity of CuO NPs AQ by MIC. The FICI at the visible MIC of the combination between CuO NPs (250 µg/mL) and AQ (15.6 µg/mL) was 0.375, clearly showing the synergistic activity of CuO NPs and AQ.

3.3. Plausible Antibacterial Mechanism

3.3.1. Biofilm Inhibition Assay

ATCC 25923 is a widely studied S. aureus strain to evaluate antibacterial and biofilm formation [54]. Biofilm formation is a potential factor that acts as a barrier against various antimicrobial agents and modifies the susceptibility pattern of S. aureus [55]. In the checkerboard assay (Figure 6), the antibacterial activity of individual CuO NPs and AQ was reduced against ATCC 25923. Because AQ and CuO NPs are correlated to antibiofilm activity [32,38], this combination will reduce the ability of S. aureus to form a biofilm structure. Moreover, a previous study on CuO NPs [56] and plant-derived anthraquinones, such as rhein and emodin, have shown potent antibiofilm activity against various Gram-negative and Gram-positive pathogens [57,58]. However, the synergistic role of AQ with a potential antibacterial metallic NPs counterpart has not been characterized. To verify this hypothesis, a CV assay was performed with the ATCC 25923 strain using CuO NPs, AQ, and CuO NPs/AQ synergistic combination. Results showed that individual CuO NPs and AQ inhibited the biofilm formation of ATCC 25923 in a dose-dependent manner, and the better ability of AQ in this action (Figure 7a and Figure S2). As expected, the inhibition activity of biofilm formation by a ½ synergistic concentration of CuO NPs and AQ (125 and 7.8 µg/mL, respectively) was stronger than those of individual materials. Quantitatively, CuO NPs at 1/8 MIC and AQ at 1/16 MIC inhibited 27% and 19% of biofilm inhibition, whereas the synergistic combination inhibited the biofilm formation of S. aureus by >80%. This highlighted that the antibiofilm activity of synergistic combination is a major antibacterial action mechanism.
In addition to CV-based biofilm inhibition experiments (Figure 7a and Figure S2), fluorescence microscopy analysis was conducted to examine the biofilm architecture of live cells because CV analysis often reflects the mixture of biofilm mass from live and dead cells [34,59]. In Figure 7b, the population of live bacterial biofilm cells after treatment of CuO NPs 1/8 or AQ 1/16 MIC was discernable compared to the control, whereas CuO NPs/AQ at 1/2 synergistic concentration displayed ~81% decrease in the total biofilm cells. Additional SEM image analysis confirmed the decreased number of live biofilm cells in the CuO NPs/AQ combination (Figure S3). All the above data demonstrated that the enhanced killing effect of CuO NPs/AQ against S. aureus is associated with biofilm architecture.

3.3.2. Morphological Characterization of Cells: Cell Shrinkage and Loss of Cytoplasmic Volume

The synergistic combination potentiates the antibacterial activity against S. aureus and predominately affects the biofilm mode of its growth (Figure 7). In general, the antibacterial activity of NPs involves membrane disruption. Therefore, the direct evidence of morphological changes or damage by synergistic materials as an additional mechanism of action needs to be investigated. To this end, SEM image analysis was performed to evaluate the physical and morphological changes in bacterial cells by the synergistic combination. In Figure 8a, ATCC 25923 cells without treating CuO NPs and/or AQ did not change their compact morphology. However, the treatment of CuO NPs or AQ at the MIC level, even for a short time, induced the irregularity and heterogeneous morphology of the cells (Figure 8b,c). This morphology was much stronger in synergistic conditions, where the severely shrunken structure of cell walls with many scars is shown (Figure 8d), confirming that cell wall deformation through the loss of cytoplasmic volume of cells by the CuO NPs/AQ synergy would enhance cell wall deformation, as shown in graphene oxides [60]. The above results and a previous report suggested a schematic model for the action of CuO NPs/AQ synergistic combination against S. aureus (Figure 9). A similar model is well known in xerotolerant bacteria, extremophiles that can survive in environments with extremely limited water availability [61]. Therefore, CuO NPs/AQ seems to make the desiccated status of S. aureus.

3.3.3. Mechanistic Behavior of the Synergistic Combination with Antibiotics

To understand the intrinsic factors or pathways involved in the synergistic combination of CuO NPs and AQ, screenings of synergistic antibiotics of known mechanisms for CuO NPs/AQ combination were performed. To this end, the MIC values for individual antibiotics by CuO NPs, AQ, or both at the sub-MIC level of CuO NPs (500 µg/mL), AQ (62.5 µg/mL), and CuO NPs/AQ (1/2 synergy; 125 and 7.8 µg/mL for CuO NPs and AQ, respectively) against ATCC 25923 cells were determined using a premade MIC plate for Gram-positive bacteria, coated with different concentrations of 19 commercial antibiotics or combinations. From the screenings (Table 3), only gentamicin (GEN) and streptomycin (STR) showed increased antibacterial activity by the synergy of CuO NPs and AQ with more than twofold, in which MIC was changed from 0.25 to <0.12 µg/mL for GEN and 2 to <1 µg/mL for STR to the control (Figure S4). To identify the origin of synergistic action, the MIC of GEN and STR was determined under CuO NPs or AQ at the 1/2 MIC level. When the MIC of GEN and STR was compared to the control and synergy samples, CuO NPs made cells more susceptible to both antibiotics, whereas that treated with AQ was reduced. Based on the inhibitory mechanism of GEN and STR, the increased susceptibility of GEN and STR against S. aureus, mainly by CuO NPs, is the inhibition of the protein synthesis pathway [62,63]. Therefore, the synergistic combination of CuO NPs and AQ might create cell shrinkage from within (Figure 6) rather than the modulation of intracellular pathways.

4. Limitations and Perspectives

CuO NPs have been suggested as a potential synergistic material in medical healthcare systems. However, its high MIC and low selectiveness against pathogens limit its use as a biocompatible antibacterial agent. In particular, the high dosage of CuO NPs against target hosts increases the chances of cell cytotoxicity, resulting in increased oxidative damage, inhibition of antioxidant pathways, chromosomal aberrations, and DNA damage. Generally, the route of synthesis, crystallographic appearance, and chemical composition affect the function of antibacterial NPs. However, CuO NPs with desired morphologies prepared with the help of physical and chemical stimuli, such as high temperature, pressure, longer reaction time, and use of different chemical reagents, have not resolved the limitation. Still, it is possible that surface modification involving chemical stability, surface reactivity, nanofabrication with capping agents, dissolution factor, and effective exposure route will enhance the biocompatible effectivity of CuO NPs, which needs to be studied further.

5. Conclusions

In this study, CuO NPs and AQ could be a selective synergistic combination against S. aureus among other Staphylococcus spp. From mechanistic studies, the combination can potentiate the antibacterial activity mainly by cell disruption and decreased biofilm formation. This study highlighted the potential effects of the synergistic combination in reducing live biofilm-forming cells. This is an additional advantage in using these combinations against recurrent infection models, such as chronic nonhealing wounds, which display a biofilm-forming cell-associated reduction in antimicrobial activity. Synergistic actions of metallic NPs with natural compounds have been reported, but the one that potentiates activity and selectiveness by CuO NPs has not been characterized. To the authors’ knowledge, this is the first study that showed the induced antibacterial capability of CuO NPs with the combination of AQ and their possible action mechanisms against S. aureus. Therefore, all information can be used in the appropriate formulation of prospective synergistic candidates that could significantly boost the use of natural antimicrobials against S. aureus.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jcs7040135/s1, Figure S1: Broth microdilution analysis using resazurin based assay against S. aureus.; Figure S2: Image of Crystal violet assay.; Figure S3: SEM analysis of S. aureus (ATCC 25923) biofilm architecture.; Figure S4: Screening of synergistic antibiotics with CuO NP, AQ, and CuO NP/AQ.

Author Contributions

P.S. and K.-s.K. designed the experiments. P.S., Y.K., and H.C. carried out the experiments. P.S. and K.-s.K. wrote the original manuscript. K.-s.K. supervised the research and revised the original manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (grant number NRF-2021R1A2C1007413).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We would like to thank the Korean Collection for Type Cultures (KCTC) for providing the pathogens (KCTC No. 13171) used in this study. Figure 1 and graphical abstract were prepared by BioRender (www.biorender.com; accessed on 13 March 2023).

Conflicts of Interest

The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.

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Figure 1. Schematic representation of CuO NPs synthesis.
Figure 1. Schematic representation of CuO NPs synthesis.
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Figure 2. XRD pattern of CuO NPs.
Figure 2. XRD pattern of CuO NPs.
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Figure 3. Morphology of CuO NPs: (a) TEM image and (b) HR-TEM image of CuO NPs.
Figure 3. Morphology of CuO NPs: (a) TEM image and (b) HR-TEM image of CuO NPs.
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Figure 4. Measurement of the (a) zeta potential and (b) size of CuO NPs. The zeta potential of both original samples from the synthesis and 1 month after synthesis was measured. Data are shown as the mean ± SD (n = 3; p < 0.05).
Figure 4. Measurement of the (a) zeta potential and (b) size of CuO NPs. The zeta potential of both original samples from the synthesis and 1 month after synthesis was measured. Data are shown as the mean ± SD (n = 3; p < 0.05).
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Figure 5. MIC of (a) CuO NPs and (b) AQ incubated against the S. aureus (ATCC 25923) strain at 37 °C for 18 h. The resazurin plate images were captured using a digital NX-200 camera (Samsung). One of the representatives is shown (n = 3). Orange circles indicate MIC values.
Figure 5. MIC of (a) CuO NPs and (b) AQ incubated against the S. aureus (ATCC 25923) strain at 37 °C for 18 h. The resazurin plate images were captured using a digital NX-200 camera (Samsung). One of the representatives is shown (n = 3). Orange circles indicate MIC values.
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Figure 6. Checkerboard analysis of CuO NPs and AQ against ATCC 25923 with resazurin dye. The 96-well plate was imaged with a digital camera (Samsung NX200). The asterisk represents MIC, and the orange circle indicates the position FICI = 0.375.
Figure 6. Checkerboard analysis of CuO NPs and AQ against ATCC 25923 with resazurin dye. The 96-well plate was imaged with a digital camera (Samsung NX200). The asterisk represents MIC, and the orange circle indicates the position FICI = 0.375.
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Figure 7. Effects of synergy on biofilm formation. (a) Relative biofilm formation (OD570/OD600) was determined for S. aureus (ATCC 25923) cells under the absence or presence of samples at MIC or half of the synergistic level. C, control; CuO NPs (1/2, 1/4, or 1/8 MIC)]; AQ (1/2, 1/4, 1/8, or 1/16 MIC); CuO NPs/AQ (1/2 synergy)]. The values in the graph represent the average ± SD (n = 7). (b) Microscopic analysis of biofilm architecture of S. aureus ATCC 25923 cells. (i) Control, (ii) CuO NPs (1/8 MIC), (iii) AQ (1/16 MIC), and (iv) CuO NPs/AQ (1/2 synergy).
Figure 7. Effects of synergy on biofilm formation. (a) Relative biofilm formation (OD570/OD600) was determined for S. aureus (ATCC 25923) cells under the absence or presence of samples at MIC or half of the synergistic level. C, control; CuO NPs (1/2, 1/4, or 1/8 MIC)]; AQ (1/2, 1/4, 1/8, or 1/16 MIC); CuO NPs/AQ (1/2 synergy)]. The values in the graph represent the average ± SD (n = 7). (b) Microscopic analysis of biofilm architecture of S. aureus ATCC 25923 cells. (i) Control, (ii) CuO NPs (1/8 MIC), (iii) AQ (1/16 MIC), and (iv) CuO NPs/AQ (1/2 synergy).
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Figure 8. Morphology changes by CuO NPs and AQ. SEM images of S. aureus ATCC 25923 cells: (a) control, (b) CuO NPs (MIC), (c) AQs (MIC), and (d) CuO NPs/AQ at the synergistic level. Overnight grown cells were treated for 3 h with samples and imaged. One of the representatives is shown (n = 3).
Figure 8. Morphology changes by CuO NPs and AQ. SEM images of S. aureus ATCC 25923 cells: (a) control, (b) CuO NPs (MIC), (c) AQs (MIC), and (d) CuO NPs/AQ at the synergistic level. Overnight grown cells were treated for 3 h with samples and imaged. One of the representatives is shown (n = 3).
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Figure 9. Schematic representation of CuO NPs/AQ combination action on the morphology of S. aureus cells.
Figure 9. Schematic representation of CuO NPs/AQ combination action on the morphology of S. aureus cells.
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Table
Table 1. MIC of CuO NPs and AQ against Staphylococcus strains.
Table 1. MIC of CuO NPs and AQ against Staphylococcus strains.
SpeciesStrain TypeStrain NumberMIC (µg/mL)
CuO NPsAQ
S. aureusType
MDR
MDR		ATCC 25923
MRSA1
MRSA2		1000
1000
1000		125
250
250
S. epidermidisType
MSSE		KCTC 13171
ATCC 12228		1000
500		125
62.5
S. saprophyticusTypeATCC 153051000250
Detailed strain information is provided in a previous report [47,48]. MSSE, methicillin-susceptible S. epidermidis.
Table
Table 2. MIC and FICI values of CuO NPs, AQ, and their combination against Staphylococcus spp.
Table 2. MIC and FICI values of CuO NPs, AQ, and their combination against Staphylococcus spp.
SpeciesStrain TypeStrain IDSingle ComponentCombination
MIC (µg/mL)FICI
CuO NPsAQCuO NPsAQ
S. aureusType
MDR
MDR		ATCC 25923
MRSA1
MRSA2		1000
1000
1000		125
250
250		250
250
500		15.6
125
62.5		0.37
0.75
0.75
S. epidermidisType
MSSE		KCTC 13171
ATCC 12228		1000
500		125
62.5		250
250		62.5
15.6		0.75
0.75
S. saprophyticusTypeATCC 1530510002505001251
Detailed strain information is provided in a previous report [47,48].
Table
Table 3. Screening of antibiotics modulated by CuO NPs, AQ, or their synergy.
Table 3. Screening of antibiotics modulated by CuO NPs, AQ, or their synergy.
AntibioticsAcronymMIC 1 (μg/mL)
Control+CuO NPs+AQSynergy
KanamycinKAN<1<1<1<1
StreptomycinSTR2<14<1
TiamulinTIA0.50.50.50.5
Quinupristin/dalfopristinSYN0.2510.250.5
LinezolidLZD2222
MupirocinMUP<0.12<0.12<0.12<0.12
VancomycinVAN1211
ChloramphenicolCHL8888
Fusidic acidFUS0.250.250.50.5
PenicillinPEN0.060.120.060.12
TrimethoprimTMP>4>42>4
RifampinRIF0.008<0.0080.0150.008
ErythromycinERY0.250.250.250.25
CiprofloxacinCIP0.120.120.250.25
TetracyclineTET0.250.50.250.5
ClindamycinCLI0.120.120.060.12
CefoxitinFOX2122
GentamicinGEN0.250.120.25<0.12
SulfamethoxazoleSMX32128>128>128
1 The MIC values were determined using the Sensititre Staphylococci antibiotic-susceptible plate (EUST2; Thermo Fisher Scientific) with a total volume of 200 μL. Red indicates the increase in the fold change compared to the control set. Reproducible data are shown (n > 3). The concentrations used for the screening were at 1/2 MIC: CuO NPs and AQ at 500 or 62.5 µg/mL, respectively, in evaluating single material with antibiotics, whereas those in the synergy were used either 125 or 7.8 µg/mL, respectively.
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Srivastava, P.; Kim, Y.; Cho, H.; Kim, K.-s. Synergistic Action between Copper Oxide (CuO) Nanoparticles and Anthraquinone-2-Carboxylic Acid (AQ) against Staphylococcus aureus. J. Compos. Sci. 2023, 7, 135. https://doi.org/10.3390/jcs7040135

AMA Style

Srivastava P, Kim Y, Cho H, Kim K-s. Synergistic Action between Copper Oxide (CuO) Nanoparticles and Anthraquinone-2-Carboxylic Acid (AQ) against Staphylococcus aureus. Journal of Composites Science. 2023; 7(4):135. https://doi.org/10.3390/jcs7040135

Chicago/Turabian Style

Srivastava, Prakhar, Yongjun Kim, Hyejin Cho, and Kwang-sun Kim. 2023. "Synergistic Action between Copper Oxide (CuO) Nanoparticles and Anthraquinone-2-Carboxylic Acid (AQ) against Staphylococcus aureus" Journal of Composites Science 7, no. 4: 135. https://doi.org/10.3390/jcs7040135

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