Calcination Temperatures, Compositions and Antimicrobial Properties of Heterostructural ZnO– CuO Nanocomposites from Calotropis Gigantea Targeted for Skin Ulcer Pathogens

An eco-friendly green route is employed for the successful synthesis of heterostructured ZnO-CuO nanocomposites using Calotropis gigantea plant and the investigation of their antimicrobial properties against skin ulcer pathogens. Binary ZnO-CuO nanocomposites prepared at calcination temperature of 300 °C exhibited superior antimicrobial effect on S. aureus, whereas the negative control sample did not show any antibacterial activities. High ZnO nanoparticles of 75 wt.% ZnO-CuO nanocomposites showed zero count of Staphylococcus aureus at a minimum inhibitory concentration of 0.625 mg/mL and minimum bactericidal concentration (MBC) of 2.5 mg/mL. Interestingly, the 75 wt.% ZnO-CuO nanocomposites exhibited strong antimicrobial activity against multi-drug resistant pathogens, with MBC ranging from 0.3125 mg/mL to 1.25 mg/mL. A time-kill assay captured a reduction in viable count from 4.3 log 10 to 1.3 log 10 after 12 h of incubation for S. aureus . Elucidating the antimicrobial activities could be useful for identifying novel ways to incorporate ZnO-CuO nanocomposites in polymers for applications in biocide materials, such as for wound dressing. Further, molecular studies are needed to explain the underlying biocidal mechanism of ZnO-CuO nanocomposites especially in the presence of Cu 2+ and Zn 2+.


Introduction
Ulcerative skin infections arising from colonisation and development of gram-positive bacteria, gramnegative bacteria and multidrug-resistant bacteria are signi cant health care problems that seriously affect human skin. A prospective quantitative study reported on the prevalence rates of skin pressure ulcers (PUs) at 15.5% in Kuala Lumpur, Malaysia (2013) [1], 33% in Palestine (2017) [2] and 16% in Bandung, Indonesia (2017) [3]. Skin infection was found in 60 (74.0%) of the collected samples from the PUs of hospitalised patients and was mainly constituted with Enterobacteriaceae strains (49.0%) such as Escherichia coli (E. coli), Klebsiella pneumonia (K. pneumonia), Enterobacter spp. and Proteus spp.; followed by Staphylococcus aureus (S. aureus) (28.0%) and non-fermenting GNB (23.0%), mostly Pseudomonas aeruginosa (P. aeruginosa), Acinetobacter spp. and Methicillin-resistant S. aureus (MRSA) [4,5,6,7]. PUs are open infected wounds that develop on the skin as result of pressure on one spot of the body for too long or from friction on the skin. Findings of new inorganic oxide antimicrobial agents synthesised from natural plant became one of the remarkable alternatives for infectious skin treatments of PUs as such options are rich in numerous varieties of metal oxides that release ions and in reactive oxygen species (ROS), such as hydroxyl radical (•OH -) and superoxide (•O 2− ) which cause increased permeability, rupture and cell death of microorganisms [8,9].
Recently, the incorporation of inorganic metal and metal oxides in sponges [10], hydrogels [11,12] and bandages [13,14] have gained priority in research because of their advantages as antimicrobial agents for treating locally infected skin ulcers. Mixed inorganic metal and metal oxides are effective disinfectants because of their relatively non-toxic pro le, chemical stability and e cient antibacterial activity (Table 1). Binary antimicrobial agents (e.g. CuO and ZnO and Ag and ZnO) have been highlighted over single antimicrobial agents given the strong synergic effect of the former in eliminating bacterial colonies at low concentration [10,24,38], more pronounced wound healing [10], lower cytotoxicity [10], better biocompatibility [24] and improved cell viability which is safe for human application [24].
Therefore, this paper focuses on the preparation of green synthesised binary ZnO-CuO nanocomposites using Calotropis gigantea (C. gigantea) leaf extract and investigates the microbial activity of these nanocomposites upon culturing with skin ulcer pathogens such as E. coli, K. pneumonia, S. aureus, P. aeruginosa and MRSA. Further, the effect of different compositions and calcination temperatures on ZnO-CuO nanocomposites were explored with respect to their prospective antimicrobial application.

Experimental Plans
Preparation of the leaves extract and binary inorganic oxides For this present investigation, the whole plant of C. gigantea was collected from Perai Pulau Pinang, Malaysia and was identi ed by the expert of Unit Herbarium, Pusat Pengajian Sains Kajihayat USM Pulau Pinang. (Herbarium No.: 11843). In this experiment work, 5 g of C. gigantea leaves were added to 100 mL of deionized water and boiled for 60 min at temperature of 90-100°C using hot plate [39,40]. Then, 50 mL of ltered leaves extracts were taken and boiled to 60-80°C using a stirrer-heater. Binary inorganic oxides

XRD analysis
The crystal phases of ZnO-CuO nanocomposites were studied using X-ray diffraction (XRD), Bruker D8 powder diffractometer operating in re ection mode with a Cu Kα radiation (40 kV, 30 mA) diffracted beam monochromator, using a step scan mode with step size of 0.030° in the range of 10° to 90°. The crystallite size was estimated from the XRD pattern using the Scherer's Equation [1]: where K= 0.9 is the shape factor, λ is the X-ray wavelength of Cu Kα radiation (0.1541 nm), θ is the Bragg diffraction angle, and β is the FWHM of the respective diffraction peak.

Time-kill assay
The antibacterial activity of ZnO-CuO nanocomposites against time was carried out using time-kill assay as illustrated in protocol before [41]. The adjusted S. aureus bacterial suspension to 0.5 McFarland standard turbidity was used and diluted with samples solution with nal concentration of 2.5 mg/mL.

Results And Discussions
Calcination temperatures and composition of heterostructural ZnO-CuO nanocomposites Seven characteristic peaks of ZnO and ve characteristic peaks of CuO were found in green ZnO-CuO samples prepared at different calcination temperatures (300, 400 and 500 °C) as shown in Figure 1. The antimicrobial e cacy of binary 50ZnO/50CuO nanocomposites at three different calcination temperatures (300°C, 400°C and 500°C) were initially characterised by S. aureus minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC) as presented in Table 2. The MIC of 50ZnO/50CuO-300C and 50ZnO/50CuO-400C for S. aureus were 2.5 mg/mL, except for 50ZnO/50CuO-500C at 5 mg/mL. Moreover, the MBC of all green synthesised 50ZnO/50CuO samples for S. aureus was at 20 mg/mL. S. aureus colonies were counted less at a concentration of 5 mg/mL for the sample 50ZnO/50CuO-300C prepared at low calcination temperature (300°C) relative to the 50ZnO/50CuO-400C and 50ZnO/50CuO-500C samples ( Figure S1). The effect of smaller sized nanoparticles generated at low (2011) veri ed that smaller particle sizes mean greater e cacy in inhibiting bacterial growth, a feature that is possibly associated with the larger surface areas of nanoparticles [50, 51].
Next, further characterisation of the differential ratios of binary ZnO and CuO nanocomposites at a calcination temperature of 300 °C were presented in Figure S2 and Table 2. The MIC of 25ZnO/75CuO-300C, 50ZnO/50CuO-300C and 75ZnO/25CuO-300C were 5 mg/mL, 2.5 mg/mL and 0.625 mg/mL for S. aureus, respectively. Similar to the MIC values, the 25ZnO/75CuO-300C and 50ZnO/50CuO-300C green samples had MBC values of 20 mg/mL and the counterpart for the 75ZnO/25CuO-300C sample was 2.5 mg/mL for S. aureus. The 75ZnO/25CuO-300C sample exerted a higher bactericidal effect against the S. aureus strain at the lowest MIC/MBC values (0.625 mg/mL/2.5 mg/mL). The antimicrobial activity was further enhanced by increasing the amount of ZnO nanoparticles in the binary compound (ZnO-CuO). The phenomenon observed can be explained by the fact that the binary 75ZnO/25CuO-300C nanocomposites are highly diffusible and generate more Zn 2+ [19]. Moreover, Cu 2+ ions bind the cell wall of host cells through surface proteins and enter the cell [19]. Subsequently, the change in the metabolism of cells leads to the microbe's cell death [19].
Further antimicrobial analysis of 75ZnO/25CuO-300C on selected skin ulcer pathogens are shown in Table 3. These pathogens are commonly associated with skin ulcer disease [4,5,6,7]. The MIC values of the green synthesised ZnO-400C for E. coli, P. aeruginosa, K. pneumonia and MRSA were at 0.3125, 0.15625, 0.625 and 0.15625 mg/mL, respectively. By contrast, the MBC values were 2.5, 0.3125, 1.25 and 0.3125 mg/mL, respectively. Furthermore, the MIC amounts for the 75ZnO/25CuO-300C sample were 0.625, 0.15625, 0.625 and 0.15625 mg/mL for E. coli, P. aeruginosa, K. pneumonia 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. The tolerance level according to the MBC/MIC ratio showed that all tested microbes are sensitive to bactericidal agents except for the CuO-500C sample against E. coli, P. aeruginosa and MRSA and the ZnO-400C sample towards E. coli. Table 3 indicates that for all tested microbes, only the tolerance levels for 75ZnO/25CuO-300C sample were less than 4, and these values identi es the sample as a strong bactericidal agent relative to other samples (ZnO-400C and CuO-500C).
Moreover, higher MBC values of the CuO-500C sample against all tested microbes possibly transpire from the slow Cu 2+ ion release from CuO nanoparticles [52]. Clearly, the ZnO-400C and 75ZnO/25CuO-300C samples show very promising results against all tested microbes. That outcome may arise from the ZnO nanoparticle's larger surface to volume ratio and the penetration of the cell membrane of the bacteria by its ions. Furthermore, the ZnO-400C sample showed better antimicrobial activity relative to the CuO-500C counterpart at a similar concentration. Some studies reported that the antimicrobial effectiveness of green synthesised inorganic oxide nanoparticles depends on particle dosage, size and treatment condition, such as calcination temperatures. This situation could be the one of the reasons for the higher antimicrobial activities of ZnO and ZnO-CuO over that of CuO particles.
Additionally, the antimicrobial activities of the ZnO-400C and 75ZnO/25CuO-300C samples are much better than that of the CuO-500C sample alone towards multi-drug resistant strains P. aeruginosa, K. pneumonia and MRSA. That outcome is obviously due to the high diffusion of Zn 2+ ions in the medium. The poor activity of CuO particles within a shorter duration suggested that the time requirement for water diffusion and subsequent Cu 2+ release in uence e cacy. The attacking delay was also associated with the cell walls of gram-negative strains. Studies have reported that gram-negative bacterial strains exhibit higher resistance or tolerance against nanomaterials compared with gram-positive bacteria [53] because of the lipopolysaccharide situated in the outer membrane of the former [54]. The cytoplasmic membrane, which is inherent to gram-negative bacteria, signi cantly maintains cellular viability. Hence, gramnegative microbes are not readily attacked by free radicals or Cu 2+ . More time and concentrated Cu 2+ ions are thus required to effectively decompose the cell membrane of the bacteria. The antimicrobial activity of ZnO, CuO and ZnO-CuO nanoparticles is due to the electrostatic interaction between positively charged zinc and copper ions (Zn 2+ and Cu 2+ ) and negatively charged microbial cell membranes [21]. In addition, the antimicrobial activity of inorganic oxide nanoparticles relies on the generation of ROS as well [17,19].
In the time-kill assay results were presented in terms of the changes in the log 10 CFU/mL of viable S. aureus colonies and indicated that the green synthesised binary 75ZnO/25CuO-300C sample exhibited signi cant bactericidal activity. The outcomes of the time-kill assay were captured in Figure S3. Figure 3 presents the time-kill curve graph for the strain. A reduction in viable count from 4.3 log 10 to 3.4 log 10 was captured after 6 h of incubation for S. aureus. By 12 h, only 1.3 log 10 of bacterial colonies were seen. At 24 h, the bacteria were completely killed. Therefore, the effective control of gram-positive S.

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Availability of data and materials
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.

Competing interests
The authors declare no con ict of interest.