Synthesis of copper oxide nanoparticles using Aloe vera leaf extract and their antibacterial activity against methicillin-resistant Staphylococcus aureus and multi-drug resistant Klebsiella pneumoniae

The global interest in nanotechnology has significantly increased in recent years as a necessity to create new and natural antibacterial agents required in the management of superbug pathogens. The present study characterized the green-synthesized copper nanoparticles derived from Aloe vera leaf extract and determined their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant (MDR) Klebsiella pneumoniae. The green synthesized copper oxide nanoparticle (CuO NPs) from the aqueous extract of Aloe vera leaves were characterized using ultra violet (UV)-visible spectra, Transmission Electron Microscopy, Fourier transform infrared, and X-ray diffraction (XRD) and then assayed for antibacterial activity. The study revealed that green synthesized CuO NPs contained several organic functional groups of phytochemicals with a wave by CuO NPs plasmon resonance. CuO NPs exhibited a plate-like hexagonal structure with a length of approximately 500 nm. The CuO NPs exhibited broad-spectrum antibacterial activity that was concentration-dependent, exhibited lower MIC and MBC values on MRSA strains (31.25–62.5 μg/mL and 125 μg/mL) than against MDR Klebsiella pneumoniae strains (62.5 μg/mL and 500 μg/mL) respectively. The CuO NPs synthesized from Aloe vera had strong bactericidal activity, offering a promising alternative strategy in the global effort to combat multidrug resistant bacterial infection. There is a need to conduct in vivo efficacy of the CuO NPs to validate their safety for human use.

Multidrug-resistant (MDR) bacteria have caused an upsurge in infectious diseases, which are now the leading cause of death (Lai et al., 2022). Antibiotic overuse and abuse are frequently the main contributors to the advancement of multidrug-resistant bacteria, which have various negative effects, including an increase in rates of mortality and morbidity, a lengthening of hospital stays, and financial loss (Hasanpour et al., 2023).

Globally, 84% of Klebsiella pneumoniae (Klebsiella pneumoniae) infections have been reported (Ferreira et al., 2019) While 55.9% have been documented in developing nations (Huynh et al., 2020). In Africa, the recurrent outbreaks of healthcare infections from K. pneumoniae have increased to an estimated rate of between 3% and 15% as compared with 7.1% among European countries (Fraser et al., 2021). Moreover, research done in Uganda has reported similar findings of 18.2% prevalence in the Eastern region (Obakiro et al., 2021), while in Southwestern Uganda, rates of 36%, 12.57% and 11.6% have been documented in Kabale district (Baguma et al., 2022), Mbarara district (Turugurwa et al., 2019) and the Bushenyi district (Odoki et al., 2019), respectively. In Uganda, the nationwide prevalence of 23.3% carbapenem-resistant (CR) and 46.3% pathogenic K. pneumoniae detected by capsular typing was reported, of which 44.1% was registered in Bushenyi district (Ssekatawa et al., 2021). Still, Baguma et al. (2022) reported 89% prevalence of extended-spectrum β-lactamase (ESBL) producing K. pneumoniae at Kabale regional referral hospital, Southwestern Uganda.

Staphylococcus aureus (S. aureus) is a gram-positive bacterium within the Staphylococci family, measuring about 1 μm in diameter, a coagulase-positive bacterium that produces grape-like cell arrangement (Maddiboyina et al., 2023). Staphylococcus aureus is a disseminated gram-positive bacterium that invades the skin, skin glands, nares, nasal epithelial cells, mucous membranes, stomach, and perineum. It regularly infects people’s skin, soft tissues, and bloodstreams. Bacteremia has a mortality rate of between 15% and 60% (Li et al., 2021). Infections with methicillin-resistant Staphylococcus aureus (MRSA), which have been shown to increase mortality and place a significant economic burden on the entire world, have been linked to the outbreak of antibiotic resistance in S. aureus isolates (Hasanpour et al., 2023).

Li et al. (2021) documented a pooled prevalence of 3% (95% CI 2%–5%) MRSA across all systemic infections and 44% S. aureus among individuals with cancer. Additionally, according to research, the prevalence of MRSA is reported to be 5% in Africa, 1% in the Americas, 2% in Europe, 4% in the Western Pacific, 8% in South-east Asia, and 0% in the Eastern Mediterranean (Abubakar and Sulaiman, 2018). Infections with MRSA have been linked to ailments related to healthcare and immunosuppression in terms of frequency and severity (Soe et al., 2021). Additionally, MRSA infections have been linked to 12% death due to MRSA bloodstream infections in adult patients with cancer after 60 days, and a 43.2% overall mortality rate after 6 months, with a subsequent community-onset infection (Tibebu et al., 2021).

In the Eastern region of Uganda, 5.7% MRSA carriage was reported among children (Kateete et al., 2019), while 8 (28.6%) prevalence of MRSA nasal colonization was reported among healthcare workers at Kampala International University Teaching Hospital, Southwestern Uganda (Abimana et al., 2019). An effective alternative to vancomycin, which is the first-line therapy advised for MRSA bacteremia, is daptomycin (Li et al., 2021). Despite vancomycin resistance being demonstrated, it is still accepted as the alternate treatment for MRSA infections, making it a risk factor for mortality (Li et al., 2021).

The recurrent outbreaks of MDR infections have led to the most recent advancements in innovative drug delivery methods for the treatment of MRSA and K. pneumoniae, including the use of hydrogels, lipid particles, nanocarriers, and polymers (Nikoletic et al., 2025). Additionally, phage treatment, antimicrobial peptides, probiotics, medicinal plants, nanocomplexes, or those with diameters less than 100 nm, and nanostructures are the most frequently mentioned substitutes for traditional antibiotics (Arsene et al., 2022).

Aloe vera, or Aloe barbadensis miller, contains various bioactive compounds that contribute to its diverse therapeutic applications, including anti-inflammatory, immunostimulatory, antibacterial, antifungal, and cell growth stimulatory properties (Alghamdi et al., 2023). Studies have shown that Aloe vera aqueous extract exhibited antibacterial efficacy against several antibiotic-resistant GP bacteria, such as MRSA linked to urinary tract infections (UTIs), and also suppression of biofilm formation in Klebsiella oxytoca and MRSA (Arsene et al., 2022).

Global interest in nanotechnology has significantly increased in recent years, and nanoscale particles derived from plants, which range from 1 to 100 nm, are known as nanoparticles. Nanoparticles are documented to possess antibacterial and therapeutic usage in the treatment of several bacterial illnesses caused by MDR pathogens, for example, bacteria, viruses, and fungi (Loo et al., 2018). This activity has been attributed to their large surface area, smaller particle size to volume ratio (Ssekatawa et al., 2020; Lai et al., 2022). Green synthesis of nanoparticles from herbal extracts has more advantages as compared to synthesis from microorganisms or chemicals, since it decreases the application of advanced intricate procedures and can also meet large-scale manufacturing needs (Kumar et al., 2015). Copper has been used as an antibacterial agent for now, decades, and both inhibitory and bactericidal effects exhibited by copper oxide NPs, for example, the recently studied copper oxide nanoparticles (CuO NPs) after the advancement of nanotechnology, have been found to suppress several oral bacteria, including Streptococcus mutans, Candida albicans, S. aureus, and K. pneumoniae (Ssekatawa et al., 2020; Ma et al., 2022).

Copper nanoparticles provide a quick and environmentally beneficial substitute against resistant micro-organisms, such as K. pneumoniae (Maddiboyina et al., 2023; Khairy et al., 2024). The green synthesis approach outperforms chemical ones since it is less expensive and less harmful to the environment (Lai et al., 2022). In comparison to other biological processes, the CuO NP synthesis mediated by plant extracts has the benefit of not requiring the maintenance of cell cultures or aseptic environments pneumoniae (Ssekatawa et al., 2020). Nonetheless, the successful utilization of herbal extracts in the green synthesis of CuO NPs has been reported in several studies (Kumar et al., 2015). Despite the reported antibacterial activity of CuO NPs synthesized from Aloe vera dried leaves against susceptible strains of S. aureus and K. pneumoniae (Abed et al., 2021), there is a need to evaluate the potential efficacy of aqueous extracts from freshly harvested leaves against MDR strains, due to their high phytochemicals (reducing and capping agents), reduce impurity and cost of processing, and availability throughout the year.

The major merits of using fresh Aloe vera leaf extract over dried leaf is due to the presence of high content of labile and water-soluble phytochemicals such as polysaccharides (acemannan), polyphenols, flavonoid, vitamins, and enzymes which act as reducing, capping, and stabilizing agents during nanoparticle synthesis (Abed et al., 2021; Liknaw et al., 2025; Anju et al., 2021; Khaldoune et al., 2024; Keabadile et al., 2020; Ssekatawa et al., 2022; Nagar and Devra, 2018). These water soluble and polymeric sugar functions as natural surfactant, bio-template, solvent and stabilizer which aid in size control and prevent agglomeration of metal/metal oxide nanoparticles (Ssekatawa et al., 2022; Nagar and Devra, 2018; Siakavella et al., 2020). In addition, the presence of these phytochemicals in fresh Aloe vera leaves enhances rapid nanoparticles synthesis, thus enhancing good energy efficiency, cost effectiveness, and simple process, which improves nanoparticles purity and prevent contamination which usually occurs in dried leaves (Abed et al., 2021; Anju et al., 2021; Khaldoune et al., 2024; Keabadile et al., 2020; Ssekatawa et al., 2022; Saravanakumar et al., 2023; Naveen et al., 2022; Bhatt et al., 2025).

The ability of metallic nanoparticles to yield reactive oxygen species (ROS), which leads to oxidative stress in the biological environment, has been the main focus of research on these materials’ antibacterial capabilities (Ma et al., 2022). Furthermore, these NPs oxidize the functional groups of proteins required for cell development, and oxidative stress causes membrane integrity loss, DNA damage, and lipid oxidation. It has been shown that several chemicals, including graphene oxide/copper oxide composites, copper oxide, copper sulfate, Ag-Cu coated nanoparticles, and others, contribute to the production of ROS that causes bacterial death (Lai et al., 2022). Due to the rising worry over MDR foodborne illnesses, the discovery of a novel natural antibacterial agent is required as a substitute for cheaper management of infections caused by superbugs (Khairy et al., 2024). Hence, a need for this study to determine the antibacterial activity of green-synthesized copper (II) oxide nanoparticles derived from fresh Aloe vera leaf aqueous extract against methicillin-resistant, because most studies reported focused on the use of dried Aloe vera leaf for synthesis of copper oxides nanoparticles.

Study design

This was a laboratory experiment designed to green-synthesize CuO NPs from an aqueous extract of Aloe vera leaves and assess their antibacterial activity against MRSA and MDR K. pneumoniae. The study was carried out between July and August 2024. The green synthesis of nanoparticle from A. vera leaf extract was conducted at the Institute of Biomedical Research (IBR) laboratory, a level 2 biosecurity facility at Kampala International University, western campus (KIU-WC) while microbiological assays of the synthesized CuO NPs was conducted at Central Diagnostic laboratory (CDL) at the College of Veterinary Medicine, Animal Resources and Biosecurity (COVAB), Makerere University. The synthesized CuO NPs derived from A. vera leaf extract were characterized at a research laboratory in India.

Test bacteria

The bacterial strains of methicillin resistant S. aureus (MRSA-A001 and MRSA-A002) and multi-drug-resistant K. pneumoniae (MDR K. pneumoniae - A001 and MDR K. pneumoniae -A002) and their respective reference strains (S. aureus ATCC 29213 and Klebsiella pneumonia NCTC 9633) were obtained from the microbial bank at CDL. These were pre-enriched in brain heart infusion (BHI) broth (Oxoid, Basingstoke, United Kingdom) at 37 °C overnight and then sub-cultured on blood agar (HiMedia laboratories Pvt. Ltd., Mumbai, India) using the streak plate technique. Subsequently, initial phenotypic identification and confirmation were done by subculturing pure colonies on both Mannitol salt agar and MacConkey agar (HiMedia laboratories Pvt. Ltd., Mumbai, India) (Cheesbrough, 2006). Kirby-Bauer disk diffusion assays was used to re-screen the clinical isolates for antibiotic resistance profile using antibiotics (Clinical and Laboratory Standards Institute (CLSI), 2025); Cefotaxime (30 μg), Gentamicin (10 μg), Cefuroxime (30 μg), Meropenem (10 μg), Cefoxitin (30 μg), Erythromycin (15 μg), Ampicillin (10 μg), Cefuroxime (30 μg), Tetracycline (30 μg), Amoxicillin clavulanic acid (30 μg), Ceftriaxone (30 μg), and Ceftazidime (10 μg).

Collection of plant materials

The Aloe vera Leaves were collected from Rukararwe botanical gardens at coordinates of latitudes (-0.52396) and longitudes (30.21723) in Bushenyi district, western Uganda. The leaves were immediately transported in a cold chain to the IBR laboratory at KIU-WC. Some of the voucher specimens were deposited at Makerere University Herbarium, College of Natural Science, Makerere University, for authentication and given a voucher number of AMA01. The plant species was identified following the flora for tropical East Africa, and then was confirmed using online databases such as Plants of the World (https://powo.science.kew.org/), The World Flora Online (http://www.worldfloraonline.org), and International Plant Names Index (www.ipni.org).

Preparation of Aloe vera leaf aqueous extract

Briefly, 25 g of A. vera Leaves were completely cleaned in clean sterile water, and then finely diced. Subsequently, the finely chopped fresh leaves were boiled for 5 minutes at 80 °C in a 250 mL Erlenmeyer flask containing 100 mL of deionized water before being cooled to room temperature. The final product was filtered twice using a 0.2 µm pore-sized Whatman filter paper to trap off any leftover solids, and to obtain a fine filtrate. This was stored as a synthetic stock of CuO NPs at 4 °C (Kumar et al., 2015).

Green synthesis of copper oxide nanoparticles

The Aloe vera CuO NPs were synthesized using a method adapted from (Kumar et al., 2015). First, a solution of copper (II) sulfates was prepared by dissolving 0.499 g of anhydrous copper (II) sulfate in 200 mL of distilled water. In addition, the mixture was prepared in an Erlenmeyer flask by adding 600 mL of A. vera extract was mixed with 50 mL of a 10 mM aqueous solution of copper sulfates (99.99% pure, Aldrich). Continuous agitation of this mixture was maintained between 100 °C and 120 °C on a shaker set at 100 rpm/min. After fiercely spinning for 24 h, the reaction mixture’s deep blue color initially turned colorless, then brick red, and finally dark crimson. The resultant solution was spun using a Beckman centrifuge with a Beckman JA-17 rotor at room temperature for 10 min at 10,000 rpm to separate the sediment mixture. Afterwards, the supernatant was aliquoted off. The CuO NPs collected were washed with distilled water and dried in an air oven overnight. The resulting powder was heated in a muffle furnace at 500 °C for 3 h, and allowed to cool before placed in a glass vial, stored at 4 °C before characterization. This extract was previously reported by Manye et al. (2023), Raphael (2012) and Sadiq et al. (2022) to contain Phenols, Tannins, Alkaloids, Flavonoids, Terpenoids, Carbohydrates, and Anthraquinones.

Characterization of synthesized copper oxide nanoparticles

This was conducted following the methods previously described by Kumar et al. (2015). Briefly, X-ray Diffraction (XRD) (PANnalytical: X’Pert PRO) using Cu-Kα radiation source at 40 kV voltage and 30 mA current and scan speed of 2°/min and scan range of 3°–90°. The XRD diffraction patterns were validated using standard CuO NPs 2θ values from the International Center for Diffraction Data (ICDD) database. The average crystallite size was computed using Debye-Scherrer’s equation given below; where d is the diameter or average crystallite size, β is the full peak width at half maximum (FWHM), λ is the X-ray wavelength used (1.5406 Å), and θ is the 2θ angle of the peak. The XRD array had eleven strong peaks at 2θ values typical of and scanning electron microscopy–energy dispersive X-ray spectroscopy (SEM-EDS) (JEOL JSM-6610-LV—with Oxford energy dispersive spectroscopy) instruments were used to analyze the structural morphology and chemical composition of CuO NPs. A transmission electron microscope (TEM) (JEM-1200EX, JEOL Ltd., Japan) was used for analysis at 25,000 kV. Briefly, 3 mL of the sample was applied to the copper grid coated with carbon to create a thin film, thin enough for electron to transmit without excessive scattering, to allow visualization of crystal lattice, grain boundary, interfaces, and defects. The excess sample was scraped off with blotting paper. The thin film was retained in the grid box individually for TEM analysis. The optical characteristics of CuO NPs were investigated using Fourier transform infrared spectroscopy (FTIR) (Shimadzu FTIR Prestige 21) scan range between 4000–500 cm -1 and UV-Vis (BioLab UV-Vis) spectroscopy with double beam scanning range between 190–1100 nm.

In vitro susceptibility activity of green-synthesized copper nanoparticles derived from Aloe vera leaf extract against MRSA and MDR Klebsiella pneumoniae strains

Antibacterial activity of green-synthesized Aloe vera leaf extract CuO NPs

The agar well diffusion method was utilized to determine the antibacterial activity of the green synthesized CuO NPs against MRSA and MDR K. pneumonia e (Khairy et al., 2024). The bacterial suspension standardized to 0.5 MacFarland standard (about 1.5–2.0 × 10 8 cfu/ml) using a densitometer device (BioSan Densitometer DEN-1B, Latvia) was surface spread uniformly on Mueller-Hinton agar (MHA) (Oxoid, Thermo Fisher Scientific, Basingstoke, United Kingdom) using sterile cotton swabs. Approximately 50 µL of Aloe vera -derived CuO NPs at concentrations of 125, 250, and 500 μg/mL were added into each well of inoculated MHA plates and then left to stand for 15 min at room temperature. These plates were later incubated at 37 °C for 24 h. Gentamicin (30 mg) and 10% Dimethyl Sulfoxide (10% DMSO) were used as positive and negative controls, respectively. The formed inhibition zones were measured as a diameter using a ruler in millimeters. The experiment was conducted in duplicates.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The MIC and MBC of green-synthesized CuO NPs were performed following the method previously described with some modifications (Khairy et al., 2024; Njeru and Muema, 2020). The assay for MIC was performed in 96-well round-bottom microtiter plates using conventional broth microdilution techniques in BHI broth, whilst the MBC test was carried out on MHA plates. The synthesized CuO NPs stock solution (500 μg/mL) was diluted twice in 100 µL of BHI before being transferred to 100 µL of BHI for the MIC test. To each of the diluted wells containing CuO NPs, an equal volume of the diluted bacterial suspension at 10 6 CFU/mL was added and thoroughly mixed, thereby achieving a final inoculum concentration of 5 × 10 5 CFU/mL. Columns for a negative control (with BHI broth alone) and a positive control (both BHI broth and bacterial suspension) were included in the experiment. The microtiter plate was incubated for 18 h at 37 °C. Subsequently, to each well of the microtiter plate, 30 µL of 0.2 μg/mL p-iodonitrotetrazolium chloride (INT, Sigma-Aldrich, USA) was added, and then later incubated at 37 °C for 30 min while repeatedly checking for any color changes. The absence of bacterial growth was indicated by the blue/purple hue of the mixture, while the bacterial growth was denoted by the pink/colorless color of the mixture. The experiment was performed in triplicates. The lowest concentration of CuO NPs that prevented bacterial growth was referred to as the MIC value (color remained blue). The MBC of CuO NPs was calculated using the microtiter wells that still had the blue/purple tint. MBC was regarded as having the lowest effective concentration of CuO NPs to eradicate bacteria. Without any sign of bacterial growth in the broth, the contents of these wells will be plated out onto fresh Muller-Hinton agar plates and incubated at 37 °C for 24 h. Three duplicates of the experiment were run. The lowest concentration of NP without any discernible growth was considered the MBC.

Data analysis

The Microsoft Excel software was used for the entry of data, and then it was exported to the Statistical for Social Sciences (SPSS) software version 26 for analysis. Descriptive statistics of mean and standard error of mean inhibition zone diameters in millimetres were determined. One-way ANOVA was used to compute Tukey’s post hoc multiple comparisons of the antibacterial activities between the nanoparticle concentrations, controls, and the test bacteria. The data obtained was presented in tables and graphs. Statistical significance was considered at p-value ≤0.05.

Composition of fresh Aloe vera leaf extract

The fresh Aloe vera leaf extract was previously reported by Manye et al. (2023), Raphael (2012) and Sadiq et al. (2022) to contain Phenols, Tannins, Alkaloids, Flavonoids, Terpenoids, Carbohydrates, and Anthraquinones.

UV–visible spectra of copper nanoparticles synthesized using Aloe vera leaf aqueous extract

Figure 1 depicts UV-visible spectra of Aloe vera leaf extract and green-synthesized CuO NPs. The plant extract reveals peaks at 240, 299, 359, and 379 nm, attributed to the plant extract, while the broad peak at 359 nm could be attributed to the plasmon resonance of CuO NPs. Other peaks, 243 and 294 nm, could be assigned to the phytochemicals in the plant extract, as shown in Figure 1. The absorption peak at 240 nm may be assigned to intense π→π* transitions in aromatic rings and conjugated C=C systems associated with phenolic and anthraquinone structures (Glushchenko et al., 2021; Ray et al., 2013; Logaranjan et al., 2016; Jia and Jia, 2009). The band around 299 nm can be attributed to higher-wavelength π→π* or n→π* transitions of anthraquinone chromophores, consistent with reported Aloe chromophore peaks at 268, 295, and 352 nm for aloin (Raphael, 2012; Sadiq et al., 2022). The peak at 359–379 nm is likely due to extended conjugation in anthraquinone and phenolic pigments, as well as other natural pigments in Aloe vera, which produce strong UV absorption into the UVA region because of conjugated double bonds and phenolic hydroxyl groups (Ray et al., 2013; Jeyaram, 2021).

In addition, the strong absorption or shoulder observed in the UV region at approximately 240 and 299 nm may be attributed to interband transitions and surface-related electronic transitions in CuO nanoparticles (Siakavella et al., 2020; Ssekatawa et al., 2022; Abed et al., 2021). The peak at 359 nm is often interpreted as a surface plasmon resonance or interband feature of CuO nanoparticles (Abosede and Obiyenwa, 2022; Kerour et al., 2018; Sathiyavimal et al., 2021; Atri et al., 2023; Bhavyasree and Xavier, 2020). This peak indicates the formation of CuO nanoparticles, and its increased intensity may be associated with the surface plasmon resonance effect of the reduced and capped CuO nanoparticles. The decrease in intensity of the shoulder peak at 379 nm suggests that phenolic groups acted as reducing agents and were consumed during the synthesis of the CuO nanoparticles.

X-ray diffraction analysis of green-synthesized copper oxide nanoparticles derived from Aloe vera leaf aqueous extract

The XRD array had eleven strong peaks at 2θ values typical of CuO NPs, as shown in Figure 2. The XRD diffractogram in Figure 2 depict 2θ angle of 28.44°, 29.44°, 35.59°, 38.5°, 40.65°, 43.20°, 48.62°, 58.02°, 61.5°, 67.29°, 68.14°, and 75.5° which are attributed the (hkl) planes of (110), (100), (111), (200), (020), (−202), (202), (202), (113), (311), (220) and (204) respectively, which are in conformity with the standard ICDD (International Center for Diffraction Data) JCPDS 00–048 – 1548 characteristics peaks attributed to monoclinic CuO (tenorite). The multiple sharp peaks confirm the crystalline nature of the CuO NPs, indicating less amorphous phase. The average crystallite size (thickness) of the synthesized CuO NPs, calculated using Debye-Scherrer’s equation, is 25 ± 2.4 nm.

Fourier transform infrared spectroscopy (FTIR)

Fourier Transform Infrared spectrophotometry was used to characterize the green synthesized CuO NPs for an insight into phytochemicals involved in reducing copper ions to CuO NPs and subsequent capping. The organic functional groups of phytochemicals in plant extracts (PEs) used in green synthesis and stabilization of CuO NP were identified by Fourier transform Infrared spectroscopy (PerkinElmer Spectrum RX I Fourier transform IR system) with a wavenumber frequency ranging from 4,000 to 500 cm -1 and a resolution of 4 cm -1 (Table 1). The Fourier Transform Infrared (FTIR) spectra of the Aloe vera extract and the green-synthesized copper oxide (CuO) nanoparticles exhibit distinct bands that correspond closely with findings documented in literature regarding plant-mediated synthesis of CuO nanoparticles. The extract reveals a broad band at 2860 cm -1 attributed to aliphatic C–H, unique anthraquinone-associated C=O/C=C overtones at 2475 and 2420 cm -1, as well as carbonyl, amide, and carboxylate bands at 1086, 1129, 1305, 1435, and 1543 cm -1. Additionally, there are pronounced C–O/C–O–C polysaccharide signals observed within the 1000–1200 cm -1 range (e.g., 1068, 1129–706 cm -1), which are consistent with the presence of polyphenols, flavonoids, proteins, and sugars (particularly acemannan) in Aloe vera, mirroring the observations reported by (Nejatzadeh Barandozi and Enferadi (2012)). In the spectra of the CuO nanoparticles, these characteristics exhibit anticipated alterations: a reduction or absence of C–H (shifted and broadened from 2860 to 3050 cm -1) and carbonyl/phenolic bands (shifting from 2475 to 2359 cm -1; 2420 to 2328 cm -1, accompanied by diminished intensity), indicative of consumption during the reduction of Cu 2+; the persistence and alteration of C=O/amide and C–O bands, signifying their role as adsorption capping agents; the emergence of a new Cu–O vibration at 640 cm -1 (within the 505–608 cm -1 range typical for monoclinic CuO); and the presence of atmospheric CO 2 artifacts in the 2300–2350 cm -1 range (Priya et al., 2023; Veisi et al., 2021; Khandelwal et al., 2024; MubarakAli et al., 2015; Bhavyasree and Xavier, 2020; Atri et al., 2023; Gebrie et al., 2025). These spectral modifications substantiate the assertion that Aloe vera polysaccharides, phenolics, and proteins function as efficacious reducing and stabilizing agents, facilitating the production of phase-pure CuO nanoparticles adorned with biomolecular capping layers, as supported by prior investigations on various plant systems in their efficacy against multidrug-resistant organisms (Saif et al., 2016; Radulescu et al., 2025; Shon et al., 2025).

Transmission electron microscopy analysis of CuO NPs derived from Aloe vera leaf aqueous extract

The Transmission Electron Microscopy (TEM) Image in Figure 3 depicts a plate-like/faceted hexagonal structure with a length of approximately 500 nm which indicated the polycrystalline nature of the synthesized nanoparticles. This gives the CuO NPs a higher surface area and reactivity due to exposed CuO NPs sites on the surface (Figure 3).

EDS elemental spectra of green-synthesized copper oxide nanoparticles derived from Aloe vera leaf aqueous extract

The EDS spectra in Figure 4; Table 2 reveal the major elements in the synthesized CuO NPs from Aloe vera extract to be Copper and Oxygen at percentages by weight of 58.37% and 41.63% respectively. This agrees with the chemical composition for copper (II) oxide.

Antibacterial activity of green-synthesized copper oxide nanoparticles derived from Aloe vera leaf aqueous extract against MRSA and MDR Klebsiella pneumoniae strains

Antibiotic resistance profile of test bacteria

From Table 3, the test isolates were screened for the occurrence of phenotypic antibiotic resistance as previously confirmed with the detected resistance genes using PCR. The isolates used in the study were previously isolated from septic wound infections; MDR K. pneumoniae- A001, MRSA-A002, while MDR K. pneumoniae -A002 and MRSA-A001 were obtained from urine and nasal swab, respectively. All the above pathogens used in the study were selected based on their multidrug resistance profile exhibited upon phenotypic re-screening for resistance patterns according to guidelines by the Clinical and Laboratory Standards Institute (CLSI). K. pneumoniae- A002 exhibited phenotypic resistance even to last resort antibiotics Meropenem, while both S. aureus strains showed resistance to cefoxitin antibiotics. The above findings showed that the isolates retained their resistance genes: the tet (A) gene, the Mec A gene, and the bla CMY gene, which were previously genotyped using uniplex polymerase chain reaction.

Antibacterial activity of green-synthesized copper nanoparticles derived from Aloe vera leaf aqueous extract against MRSA and MDR Klebsiella pneumoniae strains

The CuO NPs of exhibited a slightly higher inhibitory activity against the MRSA isolates as compared to the MDR K. pneumoniae. Still, the study showed that the antibacterial activity was significantly (p ≤ 0.0001) dependent on the concentration of the NPs used, in which higher inhibitory effects were achieved at a higher concentration of 500 μg/mL compared to 250 μg/mL and 125 μg/mL. However, there was no significant difference in the antibacterial activity of NPs at 500 μg/mL and 250 μg/mL against the MDR K. pneumoniae A001 (p = 0.4266), MDR K. pneumoniae A002 (p = 0.7765), MRSA-A001 (p = 0.4266), and MRSA-A001 (p = 0.9777). The NPs at 125 μg/mL only exhibited inhibitory effects against the two strains of MRSA (A001; 10.00 ± 0.33 mm; A00212.33 ± 0.33 mm) as compared to MDR K. pneumoniae strains, which exhibited maximum resistance, and this was statistically significant (p = 0.0005). The positive control gentamicin exhibited a statistically significant (p ≤ 0.0001) inhibitory effect against the bacteria as compared to the NPs. However, there was no significant difference in the inhibitory effects exhibited between the gentamicin and the NPs against MRSA-A002, as shown in Tables 4, 5, Figure 5, Supplementary Tables S1, S2.

Minimum inhibitory concentration and minimum bactericidal concentration of green synthesized CuO NPs derived from Aloe vera leaf aqueous extract against MRSA and MDR Klebsiella pneumoniae

The CuO NPs exhibited lower MIC values (31.25 μg/mL) against MRSA-A002 as compared to MRSA-A001, MDR K. pneumoniae- A001, and MDR K. pneumoniae- A001, which required higher concentrations of 62.5 μg/mL. A lower MBC (125 μg/mL) was required against both strains of MRSA as compared to the two strains of MDR K. pneumoniae, in Table 5.

The UV-visible spectra of Aloe vera leaf extract and green-synthesized CuO NPs revealed the formation of CuO NPs due to the absorption at 359 nm, which is associated with the Nanoparticles’ plasmon resonance peak. This finding agrees with the 355 nm reported in similar study (Keabadile et al., 2020). The plant extract reveals peaks at 379 nm that could be assigned to the reducing agent in the plant extract, since this peak is seen to reduce as the plasmon peak 359 nm increases, while other peaks at 243 and 294 nm could be assigned to the phytochemicals in the plant extract, which was in agreement with findings reported in a related study done in India (Devadoss et al., 2023). The XRD diffractogram showed angles at 28.44°, 29.44°, 35.59°, 40.65°, 43.20°, 47.53°, 48.62°, 50.92°, 55.84°, 58.02°, 67.29°, 68.14°, and 92.79°, which are characteristic peaks of CuO nanoparticles. The sharp peaks indicate the formation of crystalline CuO NPs. The peaks at 28.44°, 29.44°, 35.59°, 40.65°, 43.20°, 47.53°, 48.62°, 50.92°, and 55.84° are particularly notable copper oxide peaks. The XRD results are consistent with the TEM images, and these peaks closely match the values reported by the JPCDS No. 00–012–1041 XRD (Jabeen et al., 2024). The peak at 35.59° is assigned to the (111) plane, attributed to a monoclinic structure, as previously documented (Jabeen et al., 2024). The XRD results corroborate TEM images that depicted a crystallite size of approximately 25 ± 2.4 nm and an aggregate of plate-like hexagonal structure with a length of approximately 500 nm. This gives the CuO NPs a higher surface area and reactivity due to exposed CuO NPs sites on the surface. This was consistent with the findings reported by Mali et al. (2019) and Jawad et al. (2022). While the elemental composition reveals the presence of Copper and Oxygen with weight percentages of 58.37% and 41.63% respectively. This was in agreement with previously reported findings (Alotaibi et al., 2023). The results of TEM revealed that the CuO NPs were polyhedral in shape with a size of 100 nm in length. This was in agreement with related studies that revealed NPs with cubic crystal lattice planes/Miller’s indices were identically in conformity with the standard values for copper oxide nanoparticles reported by the International Centre for Diffraction Data (ICDD) database, file number 048–1548 (Ssekatawa et al., 2022). The polyhedral shape exhibited by the CuO NPs enhanced its efficacy for the antibacterial activity.

The Fourier Transform Infrared spectrophotometry was used to characterize the green synthesized CuO NPs from crude aqueous extracts of A. vera, which provided an insight into their chemical composition, surface functional groups, and interactions with the surrounding. The present study showed that the FTIR spectra exhibited bands related to organic functional groups, confirming capping and functionalization of the CuO NPs by the phytochemicals. The organic functional groups entrapping CuO NPs were designated as per the studies done by. The FTIR spectra of A. vera green synthesized CuO NPs exhibited several absorbance bands associated with: O-H intermolecular stretching bonds of phenolic substance, O=C=O bonds of Carbon dioxide, C-H stretching vibrations of methyl functional groups of alkanes (high peak intensity), C-H bond of alkanes, C=C bond of conjugated alkene, C-O stretching for secondary alcohol bond, and C-Br stretching of alkyl halide. This finding was similarly reported in a related study that characterized green-synthesized CuO NPs from Camellia sinensis extract and Prunus africana bark extract in Uganda (Ssekatawa et al., 2022).

However, their study on CuO NPs reported IR band peaks at 2,140–1990 cm -1 and 1,250–1,020 cm -1 linked to N=C=S bonds related to isothiocyanate and C-N stretching bond of amines, respectively, which was contrary to our study findings. Numerous studies have documented related studies that reported similar findings (Nagar and Devra, 2018; Sharma et al., 2019). This could be attributed to the variations in the phytochemical compositions of the different plants studied (Arsene et al., 2022). Studies on the phytochemical composition of A. vera leaves demonstrated the presence of pentacyclic triterpenoids with terminal OH and COOH groups, phytosterols with OH, ethyl, and methyl as the main functional groups, and ferulic acid esters terminated by aromatic rings, COOH, -CHO, -CH 2 OH, -CH 3, and -COOC 2 H 5 (Ssekatawa et al., 2022). The known phytochemical constituents in A. vera leaves correlate with the FTIR analysis report on the functional groups of extract capping CuO NPs. The FTIR analysis of the CuO NPs revealed the presence of numerous spectral peaks, which were comparable with related studies that reported a similar range of 9–12 spectral peaks (Ssekatawa et al., 2022). However, the slightly higher number of spectral peaks than the extract could be attributed to the presence of capping agents that introduce new vibrational modes and functional groups that are not present in the extract, leading to the appearance of new peaks in the FTIR spectrum. Additionally, chemical transformation involving oxidation or reduction and the addition of impurities such as ions or small organic molecules onto the surface of the nanoparticles during the synthesis process could introduce new vibrational modes and functional groups that are not present in the extract (Siakavella et al., 2020; Ssekatawa et al., 2022).

The present study revealed broad-spectrum antibacterial activity of CuO NPs against both MDR K. pneumoniae and MRSA strains at very low minimum bactericidal concentrations of 31.25 μg/mL to 125 μg/mL. This could be attributed to the abundant presence of bioactive compounds within the aqueous extracts (Nandhini et al., 2022). Similar studies have highlighted that HPLC–MS/MS chromatographic analysis of Aloe vera aqueous extract revealed abundance of aloe-emodin and its derivatives (75.75%), such as aloesin, aloe-emodine-diglucoside, and 2′-p-methoxycoumaroylaloeresin (Arsene et al., 2022). Furthermore, HPLC analysis also showed the presence of Aloins, aloinosides, and 8-O-methyl-7-hydroxyaloin in the aqueous extract. Other phytochemicals documented included: cinnamic acid derivatives, anthracene compounds, and flavonoids. Several studies have documented that each of the above phytochemicals exhibits antimicrobial activity. For example, aloe-emodin-diglucoside has been reported for its antibacterial efficacy on several gram-negative bacteria, such as MDR K. pneumoniae and P. aeruginosa (Arsene et al., 2022).

Besides the antimicrobial properties reported, anthraquinone and an isomer of emodin have been documented to exhibit several pharmacological effects, such as antiviral, antiparasitic, neuroprotective, anticancer, anti-inflammatory, and hepatoprotective activities. Moreover, Aloe vera aqueous extract has been reported to rapidly destroy biofilms formed by the majority of MDR K. pneumoniae and MRSA (Arsene et al., 2022). Henceforth, its usage in food, dietary supplements, and Ayurvedic drugs has been recognized as safe yet effective (Arsene et al., 2022). Nonetheless, a study (Ssekatawa et al., 2022) revealed that the CuO NPs green-synthesized with A. vera extract are compatible with applications requiring prolonged storage. This stability was confirmed by FTIR spectroscopy, which demonstrated that their functional groups remained consistent after 6 months of storage.

Our study findings showed the efficacy of CuO NPs with inhibition zone diameters ranging from 10.33 mm to 13.0 mm, which was contrary to the 30 mm inhibition zone diameter reported in a related study that used photosynthesized CuO NPs from Camellia sinensis extract (CSE) and Prunus africana bark extract (Ssekatawa et al., 2022). This deviation could be attributed to the variation in phytochemical compositions of the two plants used in both studies. Nonetheless, our findings were comparable to those of (Ssekatawa et al., 2022), who reported MICs ranging from 30 to 125 μg/mL against the MDR bacteria, with higher activity against MDR S. aureus. In contrast, higher activity of CuO NPs against gram-negative bacteria has been reported (Andualem et al., 2020). Furthermore, our study revealed that the antibacterial activity exhibited by CuO NPs was dose-dependent and thus increased with an increase in NP concentrations. Indeed, this finding was similarly reported in related studies that worked on CuO NPs (Siakavella et al., 2020; Manye et al., 2023).

The comparable antibacterial efficacy exhibited by the CuO NPs could be attributed to their small size, which enhances their stability as compared to large-sized NPs. Still, the isolated CuO NPs exhibited a polyhedral morphology, which could increase the amount of surface energies from different exposed crystal planes, which ultimately generates more copper ions and releases ROS within a short time. Still, studies have shown that cubic-shaped CuO NPs occasionally exhibit broad-spectrum antibacterial activity (Ma et al., 2022). The antimicrobial mechanism utilized by copper-containing NPs includes the production of reactive oxygen species (ROS) that destroy cell walls and cell membranes, and then later react with proteins and DNA (Ma et al., 2022).

It should be noted that CuO NPs have received significant attention as alternatives to AgNPs in the medical field due to their unquestionable broad-spectrum bactericidal activity, in addition to their low cost, physicochemical stability, biocompatibility properties, and compatibility with other materials to form polymers (Potbhare et al., 2019). The broad-spectrum antibacterial activity exhibited by the green-synthesized copper nanoparticles derived from A. vera leaf aqueous extract provide a good alternative to develop novel antimicrobial agents for treatment of antibiotic-resistant bacteria (Ssekatawa et al., 2020).

Limitations

This study was limited by a lack of cytotoxicity data on CuO NPs to human cells and failure to confirm if the antibacterial activity is due to the release of copper ions, disruption of the bacterial cell wall/membrane by the nanoparticle itself, or purely the ROS generation.

Recommendations

The present study recommends the need to characterize the CuO NPs synthesized from organic solvent extracts, such as ethanol and methanol, in order to obtain a comparison of the best extraction solvent for future drug development. There is a need to exploit the effects of CuO NPs on other resistant bacteria other than the ones studied in this study, such as MDR Pseudomonas aeruginosa, Enterococcus spp., Streptococci spp, and Proteus spp. The manuscript explicitly recommends future work to conduct in vivo efficacy and safety experiments to understand the precise mechanism would be crucial for optimizing the drug design and predicting the likelihood of bacteria developing resistance to the new agent.