In recent years, the term multidrug-resistant (MDR) has become synonymous with bacterial infections, which have shown increased resistance to available antibiotics, requiring immediate attention [1
]. The real threat of the MDR can be realized by estimating that MDR could cause 10 million annual deaths by 2050, surpassing cancer [1
]. In addition, this threat might be exacerbated through the heavy use of antibiotics in the developing COVID-19 pandemic to keep patients away from secondary bacterial infections. To overcome above issues, researchers have attempted to identify new classes of antibiotics, chemically modify existing drugs, and use alternatives to current antibiotics, and other methods. In particular, nano-materials have emerged as promising alternatives to current antibiotics to treat infections caused by MDR pathogens. The distinctive physiochemical properties of nano-materials enable their use in numerous biomedical applications such as bacterial killing, drug delivery, bioimaging, and anticancer treatments [2
]. In particular, nano-materials can exhibit antibacterial activity either alone [3
] or in combination with conventional antibiotics to enhance their efficiency [4
Because of their antibacterial activities, various metal and metal oxide nano-particles such as ZnO, CuO, TiO2
, MgO, Cao, Ag, Au, and Cu offer effective solutions for treating infections by MDR pathogens [5
]. ZnO has been widely used by researchers because of its antibacterial activity and distinctive physiochemical properties such as high surface area-to-volume ratio [6
]. The Food and Drug Administration has already recognized ZnO as a “GRAS” (generally recognized as safe) substance (FDA, 21CFR182.8991, USA) [8
]. Moreover, ZnO preferentially targets multiple bacterial pathways rather than using the single target approach of antibiotics [9
]. This strategy makes it difficult for bacteria to develop resistance against ZnO. Silver nano-particles (Ag NPs), another metal oxide nano-particle, have been extensively used because of their antibacterial activity [10
]. From ancient times, Ag has been used as an antibacterial and antifungal agent. However, the importance of Ag NPs has recently increased because Ag NPs are less likely to result in bacterial resistance and can be used in multi-dimensional approaches to exert antibacterial activity. Moreover, Ag NPs are used in commercial (textiles, biopolymer, and coating-based products) and medical (burn wound treatment and dental work) approaches [11
Materials prepared by combining NPs and antibiotics, known as nano-antibiotics, have recently been developed to enhance the activity of antibiotics without causing the acquisition of resistance by bacteria [12
]. As potential nano-antibiotic platforms, researchers have combined Ag NPs with conventional antibiotics to enhance antibacterial activity [4
]. Similarly, ZnO has been used with antibiotics to potentiate their synergistic actions [13
]. However, combinations of Ag, ZnO, and antibiotics have not been reported. Generally, antibiotics exert antibacterial activity either by directly interacting with bacterial components or by inhibiting their biosynthesis and degradation processes. Among the currently used antibiotics, erythromycin is on the List of Essential Medicines published by World Health Organization (Geneva, Switzerland) and is considered to be the safest and most effective medicine [14
]. Thus, erythromycin is widely used against both gram-positive and gram-negative bacteria to inhibit bacterial protein synthesis [15
]. However, bacteria can resist the activity of erythromycin by reducing their interactions with erythromycin, increasing the risk of bacterial infections that negatively affect human health. Specifically, Staphylococcus aureus
) has rapidly developed resistance to erythromycin [16
]. In contrast, NPs target multiple bacterial pathways compared rather than the single targeting used by antibiotics, and thus bacteria are less prone to become resistant to NPs [18
]. Therefore, combining Ag-ZnO NPs with erythromycin may show similar antibacterial activity while reducing drug resistance. This strategy may enable the application of currently ineffective therapies against bacterial infections to be repurposed without time- and money-consuming development of a new generation of antibiotics.
Therefore, the aim of this study is to develop a new promising nano-platform of erythromycin, which selectively inhibit the growth of S. aureus cells with inhibiting a rapid increase of bacterial resistance by use of erythromycin itself. To this end, we prepared a new nano-antibiotic platform composed of Ag NP assembled with a ZnO nano-structure and combined with erythromycin (AZE) to potentiate both the specific activity against S. aureus and its MDR strains [(methicillin-resistant S. aureus (MRSA)), and the reduction of drug resistance caused by erythromycin. A ZnO nano-structure was synthesized through low-temperature solution synthesis followed by assembly with Ag NPs.
2. Materials and Methods
2.1. Synthesis of ZnO (ZO) Nano-Structure
A simple low-temperature precipitation process was used to synthesize ZnO (ZO). Briefly, 0.2 M aqueous solution of Zn(NO3)2·6H2O, (98%, Sigma-Aldrich, St. Louis, MO, USA) was prepared in a beaker with 50 mL of deionized water (DW) before continuous stirring for 15 min at room temperature. In another beaker, 1.6 M aqueous solution of sodium hydroxide (NaOH, 95%, Junsei, Tokyo, Japan) was mixed with 50 mL of DW. Then, the NaOH solution was added to the Zn(NO3)2 reaction mixture by dropwise with continuous stirring. After adding the base, a white precipitate can be seen at the bottom of beaker. The solution was stirred at 80 °C for 6 h and then transferred to an ice bath to stop the reaction. The white precipitate was collected by centrifugation. DW and ethanol were used several times to wash the product followed by drying in an air oven at 60 °C overnight.
2.2. Synthesis of Ag-ZO (AZO)
To obtain the Ag-ZO (AZO) nano-composite, 200 mg of synthesized ZO was dispersed in 20 mL of DW and ultrasonicated for 10 min. This solution was mixed with 20.0 mL of aqueous 0.05 M AgNO3 (≥99.9%, Sigma-Aldrich) with continuous stirring, after which 2.0 g polyvinylpyrrolidone (PVP-40000, Sigma-Aldrich) was added and stirred continuously for 1 h. Next, 200 µL of NaBH4 (0.1 M) solution was added to the mixture slowly by vigorous stirring, followed by stirring for 1 h at room temperature to form AZO. Centrifugation process was used to collect the solid materials before washing with DW and ethanol. At last, the samples were kept in an air oven for drying at 60 °C overnight.
2.3. Preparation of Ag-ZO-Erythromycin (AZE)
Three different techniques were applied to prepare AZE samples for antibacterial analysis. In the first technique, synthesized AZO (50 mg) and erythromycin (50 mg) were mixed in 50 mL sterilized DW and stirred overnight. The product was centrifuged and washed with DW before drying in an air oven at 60 °C overnight. This combination of AZO and erythromycin was named AZE1. In the second technique, the combinational product was named AZE2. During agar well diffusion, 10 µL of AZO (1 mg/mL) was initially added. After 1 h, 10 µL of erythromycin (1 mg/mL) was added, followed by the incubation of plates for 24 h at 37 °C. The third technique was similar to the first technique; AZO and erythromycin were mixed in the same weight ratio and stirred overnight. The product, named AZE3, was used directly to assess antibacterial activity.
2.4.1. Material Properties
X-ray diffractometer (D8 Advance with DAVINCI design X-ray diffraction unit, Bruker, Billerica, MA, USA) with a nickel-filtered Cu Kα radiation source (λ = 1.5406 Å) was used for X-ray diffraction (XRD) study to evaluate the structures of ZO and AZO. The 2θ range of 20–80° was used to collect the diffraction patterns. Furthermore, the microstructure of a representative sample of AZO was evaluated by transmission electron microscopy (TEM; Bruker Nano GmbH, Berlin, Germany) with the help of carbon-coated 300 mesh Cu grids for holding the samples. Scanning electron microscopy (SEM) analysis was performed using a VEGA3 (TESCAN, Fuveau, France). Additionally, an Axis Supra Scanning X-ray photoelectron spectroscopy (XPS) microprobe surface analysis system (Kratos Analytical, Manchester, UK) was used to assess the binding energy and chemical state of elements in a representative AZO sample by scanning from 200 to 1200 eV. The C 1s peak position at 284.5 eV was used as the binding energy reference.
2.4.2. Preparation of Bacterial Cells
Antibacterial activity was evaluated as described previously [6
] using BBLTM
Mueller-Hinton Broth (MHB, BD Biosciences, Franklin Lakes, NJ, USA) to grow the bacterial strains purchased from American Type Culture Collection (ATCC), Manassas, VA, USA: Escherichia coli
(ATCC 25922), S. aureus
(ATCC 25923), and different MRSA clinical isolates. The MRSA strains were purchased from Culture Collection of Antimicrobial Resistant Microbes (CCARM, Seoul, Korea; www.ccarm.or.kr
) and validated by two individual experiments [19
]. Initially, the MHB medium was inoculated with single bacterial colonies and incubated at 37 °C overnight. The cultivated cells were used within 30 min for the agar well diffusion assay (Section 2.4.3
) to assess the antibacterial activity of the NPs (ZO, AZO, and AZE). In another method, cells were prepared by suspending colonies in DW. The cells were diluted to an optical density of 0.5 McFarland turbidity standard using SensititreTM
Nephelometer (Thermo Fisher Scientific, Waltham, MA, USA) to determine the minimum inhibitory concentration (MIC) (Section 2.4.4
) and characterize the cell morphology (Section 2.4.5
2.4.3. Agar Well Diffusion Assay
The antibacterial activities of ZO, AZO, and AZE against E. coli, S. aureus, and clinical isolates of MRSA strains (MRSA1 to 8) were evaluated by the well-known agar well diffusion assays. Initially, 500 µL of cultured bacterial cells was mixed with 25 mL of MHB-agar and poured into sterile petri dishes (ϕ = 90 mm) for solidification. In preliminary experiments, four holes, each 6 mm in diameter, were aseptically punched through the surface with a sterile plastic rod. Next, 20 µL of ZO or AZO (5 mg/mL), erythromycin (5 mg/mL, Sigma-Aldrich), or DW was dropped into the holes on the agar plate with bacterial cells as the experimental group, positive control, and negative control, respectively. The plates were incubated for 24 h at 37 °C, and the zone of inhibition (ZOI) produced by the materials was measured with a ruler. After assessing the initial results, similar procedures were performed to prepare MHB-agar petri dishes. Next, seven holes (each 6 mm in diameter) were aseptically punched and 20 µL (1 mg/mL) of (i) ZO, (ii) AZO, (iii) AZE1, (iv) AZE2, (v) AZE3 (vi) erythromycin, and (vii) DW were added. At the end of the experiment, antibacterial activities were assessed after measuring the diameter of the ZOI around the wells by using a transparent ruler.
2.4.4. Minimum Inhibitory Concentration (MIC) of Antibacterial Activity
The bacteria used for this experiment were diluted in MHB at a ratio of 1/1000 in MHB. Samples of ZO, AZO, and AZE3 (5 mg/mL each) were prepared with DW by serial dilution to achieve concentrations of 1.56 to 200 μg/mL. Then, 10 μL of each diluted sample was inoculated to 90 μL of the targeted bacterial medium. The bacterial cells were incubated by shaking at 500 rpm for 16 h at 37 ℃, after which the MIC was determined.
2.4.5. Morphological Characterization of Bacteria
To determine the effect of AZE3 against the same bacterium except for E. coli
, the cells were prepared as described in Section 2.4.2
. AZE3 (5 mg/mL) was added to the cell suspension to final concentrations of 25 and 100 µg/mL for S. aureus
and MRSA strains, respectively. AZE3 (5 mg/mL) and erythromycin (5 mg/mL) were used at final concentrations of 25 and 0.125 (or 0.25) µg/mL, respectively. Each sample was incubated at 37 °C with a vigorous shaking for overnight. The bacterial cells were then pelleted at 12,000 rpm for 1 min by centrifugation. With 500 µL of phosphate-buffered saline (pH 7) containing 2% formaldehyde and 1% glutaraldehyde, the pellets were resuspended and then centrifuged again for 5 min. The resulting pellets were washed twice with 1 mL of DW and resuspended in the same volume of DW. To prepare SEM image analyses, a 5 µL of aliquots were collected from the suspension and deposited on a silicon wafer (5 × 5 mm in size, Namkang Hi-Tech Co., Ltd., Seongnam, Korea) and dried at room temperature. The dried wafer was examined by SEM using a VEGA3 (TESCAN, Fuveau, France), a versatile tungsten thermionic emission SEM system, according to the manufacturer’s instructions.
2.4.6. Drug Resistance Study
To induce drug resistance, S. aureus cells (106 CFU/mL) were incubated with AZE3 (50 µg/mL) or erythromycin (50 µg/mL) and diluted accordingly for eight passages. The antibacterial activity of AZE3 or erythromycin against each passage of S. aureus cells was measured and compared to determine the efficacy of the AZE3 sample.
2.4.7. Cell Viability Study (Water Soluble Tetrazolium Salt, WST Assay)
293 [HEK-293] (ATCC® CRL-1573™) (human embryonic kidney cells) were purchased from ATCC (Manassas, VA, USA) and maintained in RPMI1640 with 10% of fetal bovine serum at 37 °C in 5% CO2. A colorimetric WST assay (Ez-Cytox; Dogenbio, Seoul, Korea) was performed to evaluate the cell viability in the synthesized samples. The cells were seeded into 96-well plates at a density of 4000 cells/well and incubated for 24 h. The cells were further incubated for 24 or 48 h in the presence of ZO, AZO, or AZE3 samples at concentrations of 10 to 50 µg/mL in 0.1% dimethyl sulfoxide. The cells were then incubated with WST reagent (one-tenth of the medium volume), after which a spectrophotometric microplate reader (BMG LABTECH GmbH, Ortenber, Germany) was used to determine the amount of formazan dye formed by measuring the absorbance at 450 nm.