Bactericidal Potency of Bioderived Carbon and Calcium Decorated on Synthesized Copper Oxide Nanocomposite from Calotropis gigantea against Primitive Wound Associated Pathogens


 Commercialized antibiotics are often linked with ineffectiveness in treating chronic wound ulcer especially associated multi-drug resistant pathogens. Photocatalytic nanoparticles have become current trade attention as potent antibacterial agent. This study investigates the bactericidal potency of green synthesised CuO nanoparticles from Calotropis gigantea (C. gigantea) leaves extract at different calcination temperatures (i.e. 400°C and 500 °C) against MDR and non-MDR wound related pathogens. A well-defined oval shape CuO nanoparticles were obtained at 500 °C (CuO-500C) with present of calcium and carbon atoms were characterized using the X-ray powder diffraction, electron microscopy, and energy-dispersive X-ray spectroscopy. FTIR and UV-Vis analysis successfully confirmed phenol and carbonyl group are the stabilizing phytochemical agents. Further antimicrobial performance of CuO-500C sample was observed against non-MDR and MDR pathogens. CuO-500C sample found to markedly inhibit biofilm formation of S. aureus. A significant reduction of non-MDR Gram-positive bacterial colonies observe starts from 6 h exposure and bactericidal activity was noticed at 12 h exposure. Furthermore, constant prolong free radicals release was observed up to 30 days which might provide advantage against long-term antimicrobial application. Overall, it proves that high concentrated natural carbon and calcium wrapped green synthesised CuO nanocomposite demonstrated potential as a strong antimicrobial agent for combating pathogens associated with open wound infections especially in long- term basis.


Introduction
Open wound ulcers such as arterial ulcers, chronic ulcers, diabetic foot ulcers, diabetic infections, pressure ulcers, skin disorders, skin infections, surgical wounds, surgical infections, traumatic wounds and venous ulcers are the most common wound injuries leading to human morbidity and mortality including amputation and death [1]. Medicare cost estimates for all wound type's therapies ranged from $28.1 to $96.8 billion where the highest expenses were for surgical wounds followed by diabetic foot ulcers [1]. It is hardly treatable when the wound is infected by multidrug-resistant (MDR) and non-MDR skin pathogens [2,3,4]. Broad-spectrum antibiotics such as vancomycin, oxacillin, penicillin, cefoxitin and chloramphenicol are ineffective to control the growth of MDR skin pathogens and are not preferred in colonized open wound ulcers [5]. Besides of that, non-MDR Gram-positive Staphylococcus aureus (S. aureus) is also one of the most common skin pathogens responsible for the infectious wound ulcers [6]. Of the 137 of wound swab samples studied at University of Gondar Referral Hospital, Northwest Ethiopia, total 136 pathogens were isolated which were mainly consist of Gram-negative (57 %) and Gram-positive (43 %) [7]. Recent study showed that, roughly 23,000 deaths a year in the United States (US) and more than 33,000 in Europe recorded due to antibiotics failure in treating MDR strain infected ulcers [8].
Increasing antibiotic resistance has stimulated research addressing green synthesised copper oxide (CuO) with different morphologies as a bactericidal agent to overcome open wound ulcers ( Table 1). The rise of green synthesised nanomaterials in biomedical eld can be a genuine support in facilitating the healing process of open wound and ultimately repairing the injured tissue. Currently, highly moisturised antimicrobial dressings including those that contain of metals or metal oxides nanoparticles are used locally to manage skin ulcers infection [40,41,42,43]. It helps in accelerating the wound recovery process by killing mechanisms of metal ions release and generation of reactive oxygen species (ROS) [14]. Other than that, the calcination temperature had a large effect on physicochemical and antimicrobial properties of metal and metal oxides nanoparticles [44]. In previous studies, F.G. El Desouky et al. (2020) determined that cerium oxide (CeO 2 ) crystallite size increases when the calcination temperature is increasing till 500°C [45] and Yu et al. (2003) found that higher calcination temperature improves crystallization of the anatase titanium dioxide (TiO 2 ) and achieve highest photocatalytic activity at 700°C [46]. It is also stated by Saravanan et al. (2015) that the high calcinations temperature results in larger average crystalline size of CuO nanoparticles [47]. Moreover, Jiao et al. (2018) identi ed that poorly crystallized and small grain size of CuO and CeO 2 with high speci c surface area (SSA) were obtained for low calcining temperature [48]. Therefore, the goal of this work is to present an alternative of simple and low-cost green synthesis for the rapid one-step fabrication of highly pure and e cient CuO/C/Ca nanocomposites by aqueous extract of medicinal plant Calotropis gigantea (C. gigantea). The traditional remedies of C. gigantea medicinal plant in treating open wound ulcers was well documented [9]. The physicochemical properties of different calcined CuO samples were studied.
Additionally, the antimicrobial properties of the CuO samples were tested against non-MDR and MDR pathogens. 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. Scanning electron microscopy (SEM Fei Quanta FEG 650) and transmission electron microscopy (TEM FEI TECHNAI F20 G2) were used for the morphology and microstructure observation of the green CuO nanoparticles, respectively. Semi-quantitative analysis of nanoparticles was carried out by energy-dispersive X-ray spectroscopy (EDAX) which is equipped with SEM machine. The primary detection of CuO nanoparticles and natural compound of leaves extract such as carbonyl and phenol group were investigated using UV-Vis spectrophotometer (Varian) at room temperature in the range of 200-700 nm. The functional groups involved in green synthesis and stabilization of CuO nanoparticles were examined using FTIR spectroscopy (Perkin Elmer). The FT-IR spectra of the green samples were recorded in the range of 4000-400 cm −1 by the KBr pellet method.

Minimum inhibitory concentration/minimum bactericidal concentration
The MIC/MBC of CuO nanoparticles were de ned by broth dilution method on 96-well plates [50]. The MIC absorbance reading at 980 nm for CuO wavelengths was recorded before and after 24 h incubation. Besides, the MIC/MBC at concentration between 10 mg/mL and 20 mg/mL for commercial CuO and green CuO-500C samples were investigated under visible light condition. The tolerance levels against green CuO nanoparticles was determined by using the stated formula:

Time-kill assay
The antimicrobial performance of green CuO-500C sample against time was evaluated according to the following time-kill assay method [50]. The adjusted S. aureus bacterial suspension to 0.5 McFarland standard turbidity was used and diluted with CuO solution with nal concentration of 20 mg/mL.

Assessment of bio lm formation
The bio lm mass of S. aureus bacterial strain treated with 20 mg/mL of CuO-500C sample was evaluated by crystal violet staining assay as described by NH

Determination of photocatalytic activity
The photocatalytic activity of green synthesised CuO-500C nanoparticles was studied by degradation of methylene blue (MB) aqueous solution [52]. In brief, about 100 mg of nanoparticles was immersed in 10 ppm of 40 mL of MB and kept in dark for a while to attain an equilibrium adsorption state. Photodegradation e ciency of CuO-500C nanoparticles was evaluated under normal room temperature condition between 1 hour and 1 month. The catalytic degradation of MB was observed by measuring UV-Visible spectra at regular time intervals.
3 Results And Discussion

Characterization of the green synthesised CuO/C/Ca nanocomposite
The morphology of the green CuO nanoparticles was studied using scanning electron microscopy and transmission electron microscopy ( Fig. 1(e), Fig. 1(f) and Table 2). The SEM images (Figs. 1(a) and (c)) show that the CuO-400C sample have mixture of rod and quasi-spherical shaped nanoparticles and that CuO-500C sample are mainly oval in shape with agglomerated nano-sized morphology. A histogram showing the particle size of distribution for CuO-500C nanoparticles which mainly fall between 20 and 22 nm ( Fig. 1(h)). Figure 1(g) shows the TEM lattice fringes with distance of 0.254 nm for CuO-500C nanoparticles. It was found that rod-shaped microstructures resulted in a slight decrease in the cidal properties against S. aureus which is further explained in detail on antimicrobial investigation part ( gure S1). It is due to the reduced available SSA. Whereas, oval structures can increase the available SSA for Cu and O atoms. Some weak peaks for C, Ca, S, Cl, K and Mg atoms are seen for CuO green nanoparticles indicates the participation of C. gigantea leaves extract phytochemical compounds during the green synthesis. The CuO nanoparticles are well encapsulated by C. gigantea leaf extract ( Fig. 1(a)), and this similar phenomenon is reported in previous research, where Au nanoparticles are well dispersed in G. mangostana peel extract [53].  52761Å. Some additional X-ray diffraction peaks was observed in the powdered C. gigantea leaf sample pattern which demonstrated the CuO nanocomposite being well encapsulated and interacted with natural compounds such as carbon and calcium. These natural compounds could further accelerate antimicrobial activity of CuO nanoparticles synergistically [54,55,56]. The crystallite size of CuO nanoparticles was estimated from the XRD pattern using the Scherer's Equation at highest FWHM peak ( Table 2).
The FT-IR analysis was performed to investigate participation of phytochemical compounds in stabilising and reducing green CuO nanoparticles ( Fig. 2(b)).
A sharp peak at 3438 cm − 1 was assigned to O-H stretching polyphenols ( avonoids). The peaks between 1633 cm − 1 and 1765 cm − 1 were assigned to carbonyl group. The absorption peaks at 1385 cm − 1 was assigned to vibration mode of esters. Besides absorption peaks between 1110 and 1115 cm − 1 belongs to biomolecules of C. gigantea leaves extract [57,58]. The low reported absorption peak at 540 cm − 1 is characteristic of CuO group [59,60].
The UV-Vis absorbance spectra of C. gigantea extract and green CuO sample were illustrated in Fig. 2(c). C. gigantea extract was referred as a control solution which does show two prominent absorbance peaks in UV spectrum at 206 nm (carbonyl compounds) and 269 nm (phenolic compounds) [61].
Solution with CuO nanocomposites illustrated a sharp distinct absorbance peak at 233 nm which belongs to natural carbon [62]. The produced nanoparticles can be identi ed by the appearance of small band in their respective spectra which was around 307 nm for CuO-500C nanocomposite [57].

Minimum inhibitory concentration/minimum bactericidal concentration
The wound associated pathogens are a signi cant global public health threat with serious wound care and management incurring huge economic costs. S. aureus, E. coli, P. aeruginosa, K. pneumoniae and MRSA are the prevailing microbial pathogens that occur in patients with open colonised wounds. Grampositive S. aureus bacteria are among the most common pathogens associated with open wound infections [63]. Overall results prove that high concentrated green CuO/C/Ca nanocomposites show more enhanced biocidal activity against various wound pathogens. The preliminary MIC results of CuO-400C exhibit that CuO nanoparticles possess considerable inhibition effect on the S. aureus colony counts. The MIC for the CuO-400C is 5 mg/mL for S. aureus. At a dosage of 10 mg/mL, the result does not show any antibacterial activity ( gure S1(b)), where MBC cannot be achieved at this amount. The antimicrobial activity of CuO-500C is more pronounced than that of CuO-400C, and the MIC/MBC against S. aureus is 5 mg/mL/20 mg/mL ( gure S1). Our MIC/MBC results have shown that the CuO-400C nanoparticles produced at low annealing temperature is less effective in killing microbes at low dosage compared with the CuO-500C sample due to reduced SSA [64]. The strong antimicrobial effect of CuO-500C sample was attributed to the smaller size and the oval shape which is produced at high calcination temperature. The surface reactivity increases due to the size and the shape of the particles. Previously, Azam et al. (2012) revealed that the small CuO particles synthesised at 400°C have a zone of inhibition radii twice that of particles produced at 700°C and that the zone of inhibition decreases with increasing annealing temperature from 400°C to 700°C [44]. N. Nasihatsheno (2019) identi ed that the calcination temperature affects the size and the shape of the nal products and that the variation in the reaction temperature from 400°C to 600°C leads to different particle sizes and morphologies of CuO nanoparticles [65]. Sung Woo Oh et al. (2007) pinpoint in their research that the crystallite size of CuO increases until the calcination temperature reaches 500°C and that no signi cant change in crystallite size is observed at high calcination temperatures (> 500°C) [66].
Furthermore, the CuO samples prepared at annealing temperatures of 300°C and 400°C exhibit similar morphological features (e.g. size and shape), but those prepared at 500°C exhibit different morphological changes [66].
So, in next set of experiment the antimicrobial performance of CuO-500C sample against non-MDR Gram-negative and MDR pathogens were discussed. The tolerance level based on MBC/MIC shows that the tested strains such as E. coli, P. aeruginosa and MRSA are not likely sensitive to bactericidal agents. Table 3 illustrates the tested non-MDR and MDR strain. It is evident from Table 3, S. aureus and K. pneumoniae is more susceptible to CuO-500C nanocomposite compare to other tested pathogens. This study recommends that killing mechanisms of CuO/C/Ca nanocomposite towards different species of pathogens should be further investigated.  S2 (b)), green CuO-500C sample had a higher activity in the light rather than the dark ( gure S1 (c)), except for the commercial CuO that had no effect on bacterial reduction.
From here it can be concluded that, CuO-500C which has smaller size and uniform oval shape stand as most potential antimicrobial agent than CuO-400C sample. It also has been identi ed that the strong antimicrobial effectiveness of green CuO-500C sample under uorescent light illumination is most likely due to oxidative stress caused by ROS which was activated through photocatalytic reaction [60].

Time-kill assay
The time-dependent bactericidal activity of optimized green CuO-500C sample were determined against S. aureus. The outcomes of the experiment are shown in Figs. 3, and S3. A signi cant reduction of ≤ 2.5 log 10 in the bacterial colonies proportionally with time were observed for CuO green sample. A reduction in viable count from 4.3 log 10 to 2.5 log 10 is captured after 6 h and complete killing at 12 h. Results demonstrate that a high dosage of CuO-500C (i.e. 20 mg/mL) are required to eliminate 100% bacterial counts within 12 h. The untreated bacterial strains show no reduction in colony counts even after 24 h of incubation period. As captured in the time-kill analysis, a signi cant reduction of non-MDR Gram-positive S. aureus colonies were achieved at 6 h exposure.

Inhibition of bio lm formation
The bacterial bio lm formation was studied using method of crystal violet assay. CuO-500C sample exhibited signi cant reduction of OD when compared to the negative control sample (Fig. 4). Results showed that the CuO-500C sample was found to markedly inhibit bio lm formed by S. aureus. The antimicrobial action of green CuO nanoparticles might be generally falls within killing mechanisms from free metal oxide ions release and generation of reactive O species (ROS; •O 2− , •OH − , Cu 2+ and Ca 2+ ) [14]. Further investigation on metal ions release (Cu 2+ and Ca 2+ ) is recommended.

Kirby-Bauer disc diffusion test
The antimicrobial activities of green CuO-500C and commercial CuO samples at two different concentration (2.5 mg/mL and 10 mg/mL) and C. gigantea leaves extract were examined on S. aureus. The ZOI reveals that CuO-500C sample was effective towards Gram-positive pathogen when increasing the concentration amount from 2.5 mg/mL to 10 mg/mL. The previous MIC/MBC results agrees with the nding in this study, in which the CuO nanoparticles antimicrobial effectiveness are concentration-dependent (Table 4 and gure S4). In accordance with Kirby-Bauer disc diffusion ndings, the enhanced antimicrobial activity was seen for green CuO-500C sample at 10 mg/mL when compared with other samples. It might be attributed to rise in more free metal oxides ions and ROS at highest concentration of green nanoparticles. The photodegradation e ciency of CuO-500C nanoparticles has been studied on the degradation of MB aqueous solution (Fig. 5). The color of the MB slightly decreased after 1 hour at normal room temperature condition and a sharp absorbance peak at wavelength of 665 nm was dropped signi cantly. Our methylene blue photocatalytic experiment demonstrated that the green synthesised CuO nanoparticles were found more active for the degradation of MB in a relatively long interval of time which is about 1 month. It might be attributed to the slow and steady release of free radicals at normal room temperature condition. The performance of 73.33 % for MB color removal was reached at 1-month duration (Table 5), which indicated that natural carbon and calcium wrapped CuO-500C nanoparticles could prolong the release time of free radicals in steady manner for long-term antimicrobial application and create stable environment that might provide biocompatible advantage [67]. It's also believed that overproduced hydroxyl radicals in short duration of time has reactive and hazardous free radicals which causes disturbance to wound-healing related cells and broblast [68]. Successful control on steady and slow release of free radicals such as •O 2− and •OH − from this green synthesised nanoparticles might present with biocompatible properties towards human cells and can accelerate wound healing properties [69]. Besides of that, the green synthesised CuO-500C nanoparticles also might be a promising antibacterial photocatalytic agent for wound dressing application.

Conclusions
This study reveals the potential of bioderived carbon and calcium decorated on synthesized copper oxide nanocomposite (CuO-500C) as antimicrobial agent against non-MDR and MDR wound associated pathogens. This bactericidal agent works effectively in concentration-dependent manner and demonstrates strong bio lm inhibition properties against Gram-positive non-MDR strain. The control on steady and slow release of free radicals from this green synthesised nanoparticles might be bene cial biocompatible advantage that can accelerate wound healing properties. Further understanding on photodegradation e ciency of CuO-500C nanoparticles under irradiation of direct sun light or ultra-violet light is needed especially on ROS release (