Compositions and antimicrobial properties of binary ZnO–CuO nanocomposites encapsulated calcium and carbon from Calotropis gigantea targeted for skin pathogens

Calotropis gigantea (C. gigantea) extract with an ecofriendly nanotechnology approach could provide promising antimicrobial activity against skin pathogens. This study investigates the antimicrobial capability of green synthesized binary ZnO–CuO nanocomposites from C. gigantea against non-MDR (Staphylococcus aureus and Escherichia coli) and MDR (Klebsiella pneumoniae, Pseudomonas aeruginosa and methicillin-resistant S. aureus) skin pathogens. Scanning electron microscopy and transmission electron microscopy revealed the size and shape of B3Z1C sample. Results of X-ray powder diffraction, energy-dispersive spectroscopy, FTIR and UV–Vis spectroscopy analyses confirmed the presence of mixed nanoparticles (i.e., zinc oxide, copper oxide, carbon and calcium) and the stabilising phytochemical agents of plant (i.e., phenol and carbonyl). Antimicrobial results showed that carbon and calcium decorated binary ZnO–CuO nanocomposites with compositions of 75 wt% of ZnO and 25 wt% CuO (B3Z1C) was a strong bactericidal agent with the MBC/MIC ratio of ≤ 4 and ≤ 2 for non-MDR and MDR pathogens, respectively. A significant non-MDR zone of inhibitions were observed for BZC by Kirby–Bauer disc-diffusion test. Further time-kill observation revealed significant fourfold reduction in non-MDR pathogen viable count after 12 h study period. Further molecular studies are needed to explain the biocidal mechanism underlying B3Z1C potential.


Minimum inhibitory concentration (MIC)/MBC determination and tolerance level.
Antibacterial activity of BZC nanocomposites against S. aureus 29213, E. coli 25922, P. aeruginosa 27853, K. pneumoniae 700603, and MRSA 38591 were assessed using broth-dilution method on 96-well plates as described by Harun et al 46 . Absorbance was read at 980 nm wavelength 46 . High wavelength was selected because of BZC nanoparticle deposition. The bactericidal and bacteriostatic capacity of the samples was determined by the tolerance level 46 . Time-kill assay. The antibacterial activity of BZC nanocomposites against time was performed using timekill assay as illustrated in a previous protocol 46 . S. aureus bacterial suspension adjusted to 0.5 McFarland standard turbidity was used and diluted with sample solution to a final concentration of 2.5 mg/mL.

Kirby-Bauer disc-diffusion test.
The antibacterial activity of BZC nanocomposites against S. aureus was further evaluated using Kirby-Bauer disc-diffusion test 49 . BZC nanocomposite solutions (2.5 and 10 mg/mL) were prepared and used further for antibacterial studies. About 20 µL of BZC nanocomposite solution, negative control (10% DMSO + distilled water), and C. gigantea leaf extract were loaded into 6 mm sterile filter papers, and the solution was allowed to be diffused within 15-30 min. Then, all discs were properly placed on agar which was already previously spread with bacterial culture. A standard antibiotic comprising 10 µg of Oxoid streptomycin antimicrobial susceptibility discs served as a positive control. After 24 h of incubation at 37 °C, the different levels of zone of inhibition were measured. 17

Results and discussion
Surface morphology of binary ZnO-CuO nanocomposites. The SEM images of BZC nanocomposites are shown in Fig. 1. B1Z1C had a porous nature ( Fig. 1c) with few irregular rod-shaped particles (inset in Fig. 1c). Meanwhile, B1Z3C (Fig. 1a) and B3Z1C (Fig. 1e) had porous honeycomb structures with agglomerated morphology (inset in Fig. 1a,e). The large porous honeycomb structures further increased the available surface area for antimicrobial activity 26 . These uniform pores were produced during green synthesis owing to the escape of gases at high temperatures 26 . The EDAX profile of the green synthesised B3Z1C nanocomposites confirmed the presence of Zn, Cu, and O, which were about 49.97 wt%, 20.34 wt%, and 21.32 wt%, respectively. Some weak signals for C, Mg, S, Cl, K, Na, and Ca atoms were found for all BZC nanocomposites (Fig. 1b,d,f). Similar results have been reported for green nanoparticles derived from Artemisia haussknechtii leaf extract 50 , aqueous Artemisia haussknechtii flower extract 9 , Protoparmeliopsis muralis lichen 51 , Ochradenus baccatus leaves 52 , and Jatropha curcas L. leaf 53 . The presence of elements such as C, Mg, S, Cl, K, Na, and Ca in small amounts indicated the participation of plant phytochemical groups in reducing and capping the green synthesised BZC nanocomposites 9,50-53 . Meanwhile, the TEM image of B3Z1C nanocomposites revealed irregular oval and quasispherical shape with an average length of 8.126 nm and diameter of 7.515 nm in size (Fig. 1g). These structures could increase the available surface area for reaction. The magnified TEM image of the B3Z1C nanocomposites  (Fig. 1h).      (Fig. 2). This finding was possibly due to the presence of the phytochemical element of C. gigantea leaves as a capping and reducing agent 47 . The XRD patterns of powdered C. gigantea leaves successfully revealed trace natural elements such as calcium and carbon (Fig. 2) 55 .

Crystal analysis of binary
The main novelty of this study was the detection of pythochemical elements such natural calcium 56 and carbon 54,57 in leaf extract, which could further boost the antimicrobial activity of BZC nanocomposites. Calcium

UV-Vis spectroscopy analysis of binary ZnO-CuO nanocomposites.
The UV-Vis diffuse reflectance spectra of C. gigantea extract and B3Z1C nanocomposites are shown in Fig. 4. The appearance of a small broad peak at approximately 317 nm indicated the formation of irregular oval and quasi-spherical B3Z1C nanocomposites. Absorption peaks at 206 nm could be attributed to various chromophores, including the C=C bond of various compounds, the C=O bond of carbonyl compounds, and the benzene ring, whereas the absorption peak at 269 nm may be related to the various aromatic compounds, such as phenolics 60 . A sharp distinct peak was found at 233 nm owing to the formation of natural graphene-like carbon which played an important role in antimicrobial efficacy against MDR strains 61 .
Antimicrobial properties of binary ZnO-CuO nanocomposites. About 37% of patients with skinulcer disease are infected with Gram-positive S. aureus pathogen 62 . The antimicrobial characterisation BZC nanocomposites with different ratios is presented in Fig. S1 and Table 4. The MICs of B1Z3C, B1Z1C, and B3Z1C were 5, 2.5, and 0.625 mg/mL for S. aureus, respectively. Similar to the MIC values, B1Z3C and B1Z1C had MBCs of 20 mg/mL, and the counterpart for B3Z1C was 2.5 mg/mL for S. aureus. B3Z1C exerted a higher bactericidal effect against the S. aureus strain at the lowest MIC/MBC values (0.625 mg/mL/2.5 mg/mL). Antimicrobial activity was further enhanced by increasing the amount of ZnO nanoparticles in the binary compound (ZnO-CuO). This finding can be explained by the fact that the binary B3Z1C nanocomposites were highly diffusible and able to generate more Zn 2+ ions 19 . Moreover, Cu 2+ ions bound the cell wall of host cells through surface proteins and entered the cell 19 . Subsequently, the change in cell metabolism led to the microbe's cell death 19 . Commercial B3Z1C was also prepared and tested against S. aureus for comparison. Results showed that com-   (Table 4 and Fig. S1). However, the green B3Z1C was labelled as a strong bactericidal agent because the tolerance ratio was ≤ 4. Further antimicrobial analysis of B3Z1C nanocomposites was conducted on selected skin-ulcer pathogens, and results are shown in Table 5. These pathogens are commonly associated with skin-ulcer disease [4][5][6][7] . Also, the inhibitory activities of binary antimicrobial agents on bacterial colonies highly depend on the antimicrobial efficacy of dual-ionic systems and types of microbial pathogens, such as non-MDR Gram-positive bacteria (S. aureus), Gram-negative bacteria (E. coli) and MDR bacteria (P. aeruginosa, K. pneumoniae, and MRSA). The MIC amounts for B3Z1C were 0.625, 0.15625, 0.625, and 0.15625 mg/mL for E. coli, P. aeruginosa, K. pneumoniae, and MRSA, respectively. MBC values with 2.5, 0.3125, 1.25, and 0.3125 mg/mL were also observed for this green binary inorganic oxide sample. Table 5 indicates that for all tested microbes, the tolerance levels for B3Z1C were   www.nature.com/scientificreports/ less than 4, indicating that the sample was a strong bactericidal agent. Binary B3Z1C has strong antimicrobial activity against Gram-negative bacteria (E. coli). Table 5 is the evidence for this finding. Clearly, B3Z1C showed very promising results against all tested MDR microbes such as P. aeruginosa, K. pneumoniae, and MRSA. This outcome may be due to the B3Z1C nanoparticles' larger surface-to-volume ratio and the cell-membrane penetration of the bacteria by its ions. Some studies have reported that the antimicrobial effectiveness of green synthesised inorganic oxide nanoparticles depends on high particle dosage and small nanoparticle size, which could explain the higher antimicrobial activities of B3Z1C. The antimicrobial activity of B3Z1C was due to the electrostatic interaction between positively charged zinc and copper ions (Zn 2+ and Cu 2+ ) and negatively charged microbial cell membranes 21 . The antimicrobial activity of B3Z1C nanocomposites relied on the generation of ROS as well 17,19 . Moreover, free ions from natural organic carbon and calcium derived from C. gigantea leaf extract played an important role in exerting the synergic effect that killed MDR microbes at very low concentrations 54,56 .
Results of time-kill assay were presented in terms of the changes in log 10 CFU/mL of viable S. aureus colonies, as shown in Fig. S2. The green synthesised B3Z1C nanocomposites were found to have significant bactericidal activity. Figure 5 presents the time-kill curve graph for the strain. Generally, bacterial growth includes a log or exponential phase in which bacterial-cell doubling occur and their biomass increases from day 1 to day 2 63,64 . A reduction in viable count from 4.3 log 10 to 3.4 log 10 was observed after 6 h of incubation for S. aureus. By 12 h, only 1.3 log 10 of bacterial colonies were found. At 24 h, the bacteria were completely killed. Thus, Gram-positive S. aureus bacteria were effectively controlled by the synergistic combination of 75 wt% of ZnO and 25 wt% of CuO nanoparticles in the presence of natural graphene-like carbon, calcium, and phytochemical constituents such as cardiac glycosides, tannins, saponins, terpenes, flavonoids, and phenolics in C. gigantea leaf extract 54,56,[65][66][67][68] .
Furthermore, Kirby-Bauer disc-diffusion method was used to evaluate the antimicrobial activity of BZC nanocomposites against Gram-positive S. aureus. The cultures exposed to negative control sample did not show any inhibition zones around the filters, indicating that they did not have any antibacterial properties. However, B3Z1C exhibited a wider zone of inhibition (ZOI) than other BZC samples possibly because of the nanoparticle size and the fast diffusion of metal ions into agar medium ( Fig. S3 and Table 6). The antimicrobial activity of all green BZC samples further improved with increased concentration. C. gigantea extract also exhibited a slight ZOI toward S. aureus which could be attributed to bioactive compounds such as carbonyl and phenolic groups. The antibiotic streptomycin serving as a positive control exhibited a larger ZOI, as shown in Fig. S3 and Table 6.  Table 6. Kirby-Bauer disc diffusion ZOI (mm) of BZC nanocomposites against S. aureus. These data represent mean (± SD) of three replicates. NA symbolises no bacterial activity found in this work.

Conclusions
Binary B3Z1C nanocomposites prepared at compositions of 75 wt% of ZnO and 25 wt% CuO demonstrated significant antimicrobial property against non-MDR and MDR pathogens with tolerance ratio of ≤ 4 and ≤ 2, respectively. Besides, promising antimicrobial effect of B3Z1C sample towards non-MDR bacteria (S. aureus) were seen from disc diffusion assay and time kill analysis. The mechanisms underlying the biocidal activity of B3Z1C nanocomposites may involve the presence of natural carbon, free ions (i.e., Cu 2+ , Zn 2+ and Ca 2+ ), and ROS. Further In vitro and In vivo toxicity studies are needed to understand B3Z1C efficiency in treating PU infections.

Data availability
The datasets generated and/or analysed during the current study are not publicly available due to the patent application for methods of making and using and compositions of binary nanocomposites formed by green synthesis but are available from the corresponding author on reasonable request.