Green synthesized Ag–Cu nanocomposites as an improved strategy to fight multidrug-resistant bacteria by inhibition of biofilm formation: In vitro and in silico assessment study

Jafar Abdulkareem Abbas; Buthenia Abd Alhamza Hasoon; Majid Sakhi Jabir; Suresh Ghotekar; Ayman Abdel-Aziz Swelum

doi:10.1515/gps-2025-0009

Article Open Access

Green synthesized Ag–Cu nanocomposites as an improved strategy to fight multidrug-resistant bacteria by inhibition of biofilm formation: In vitro and in silico assessment study

Jafar Abdulkareem Abbas , Buthenia Abd Alhamza Hasoon , Majid Sakhi Jabir , Suresh Ghotekar and Ayman Abdel-Aziz Swelum

Published/Copyright: July 23, 2025

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From the journal Green Processing and Synthesis Volume 14 Issue 1

Abstract

This study explores the green synthesis and characterization of monometallic Ag nanoparticles (NPs), Cu NPs, and bimetallic Ag–Cu NPs mediated by the aqueous extract of Syzygium aromaticum as a reducing agent. It aims to address the growing problem of antibiotic-resistant bacterial infections, particularly those caused by Escherichia coli and Staphylococcus saprophyticus. The synthesized NPs were characterized using techniques such as UV-Vis, X-ray diffraction, and transmission electron microscopy, and their antimicrobial, antibiofilm, and ROS production activities were evaluated. Results revealed that bimetallic Ag–Cu NPs exhibited higher antibacterial and antibiofilm effects than monometallic counterparts, with enhanced synergistic effects when combined with antibiotics. Because of the importance of Ag NPs and Cu NPs, the application of molecular docking simulations to Ag NPs and Cu NPs can provide comprehensive insights into their chemical and biological properties. This technique collectively aids in assessing the viability of Ag NPs and Cu NPs as drug candidates, optimizing their structures for better efficacy and safety, and predicting their behavior within biological systems. The study concludes that Ag–Cu NPs hold promise for addressing resistant bacterial strains and biofilm-related infections.

Keywords: Ag-Cu NPs; antibacterial; antibiofilm; MDR; ROS; in silico study

1 Introduction

Bacterial infections exhibiting antibiotic resistance, mostly nosocomial, remain a significant issue in the early twenty-first century owing to antibiotic abuse, inadequate cleaning practices in hospitals, and insufficiently understood mechanisms to evade unnecessary treatment. The fast increase in bacterial resistance necessitates the exploration of novel techniques, and the use of nanomaterials could be a useful option. Currently, the integration of antibacterial polymers with nanoparticles (NPs) and bioactive agents has been evaluated to enhance biocidal treatment [1,2,3]. Metal NPs, especially silver (Ag) and copper (Cu), have emerged as promising alternatives due to their multimodal antibacterial actions: (i) membrane disruption, (ii) reactive oxygen species (ROS) generation, and (iii) enzyme inhibition. Unlike antibiotics, NPs target multiple cellular pathways simultaneously, reducing the likelihood of resistance development. However, monometallic NPs often require high doses for biofilm penetration, risking cytotoxicity [4,5,6]. Bimetallic NP synthesis involves the sequential or simultaneous reduction of two distinct metals to create either an alloy or a core–shell nanostructure. The process where a shell metal is reduced onto a pre-formed core metal seed is sequential reduction. This approach presents a compelling treatment option due to its potential applications [7]. Pollution control requires innovative, economical, and environmentally beneficial approaches to chemical processes, which must be developed in response to global needs. The low long-term cost of environmentally friendly procedures is the reason for the green synthesis expansion. There has been a tendency toward the development of low-tech, environmentally friendly methods for producing bimetallic alloyed NPs as competitive alternatives. Here, a simple one-pot reaction method was developed to create homogenous Cu/Ag alloy nanostructures [8]. Silver nanoparticles (AgNPs) exhibit antibacterial, antiviral, and antifungal properties. Research indicates that plant-synthesized AgNPs possess potent antibacterial and antibiofilm activities, making them effective against various clinically relevant infections. In contrast, copper nanoparticles (CuNPs) are well known for their strong antibacterial properties and have been utilized in storing drinking water since ancient times. CuNPs demonstrate significant antibacterial and antifungal effects against a variety of harmful bacteria and fungi, as well as sufficient cytotoxicity against bacterial strains such as S. saprophyticus and E. coli. While Ag–Cu NPs work synergistically to target both Gram-positive and Gram-negative bacteria, pathogenic E. coli are facultative pathogens that are normally found in the human intestinal flora. Infections associated with E. coli include hemolytic-uremic syndrome, meningitis, septicemia, surgical site infections, urinary tract infections (UTIs), and hospital-acquired pneumonia (HAP) [9,10]. Also, Staphylococcus bacteria are related to human infections. Clustered Gram-positive Commensal CoNS S. saprophyticus is a common urinary tract pathogen that causes 10–20% of cases in young women. It is seldom associated with UTIs in male populations [11]. Employing their virulent component, such as biofilm creation that both bacteria are widely known for their ability to produce biofilms, which are cell communities that may be used for invasion, pathogenicity, and resistance on a variety of surfaces. A three-dimensional structure known as microcolonies or mature biofilm fields, in which cells are embedded in the extracellular matrix, mostly composed of proteins, DNA, and polysaccharides, is indicative of a biofilm [12]. Here, we report a one-pot green synthesis of Ag–Cu NPs using Syzygium aromaticum (clove) bud extract as a reducing and stabilizing agent. Clove phytochemicals (e.g., eugenol, gallic acid) mediate the NP formation. They are then characterized by UV-Vis, X-ray diffraction (XRD), and transmission electron microscopy (TEM) techniques; the biosynthesized NPs were evaluated physicochemically. Likewise, Gram-negative bacteria E. coli and Gram-positive bacteria S. saprophyticus were employed to verify the antimicrobial antibiofilm activity and evaluate synergistic influence with various kinds of antibiotics for each type of NP described earlier. To the best of our knowledge, this is the first study to compare and assess the antibiofilm efficacy and ROS-mediated killing via H₂DCF-DA of Ag, Cu, and Ag–Cu NPs synthesized using Syzgium aromaticum against E. coli and S. saprophyticus.

2 Materials and methods

2.1 Plant extraction and synthesis of Ag, Cu, and Ag–Cu NPs

Tender flower buds of the Syzgium aromaticum plant were collected from the agriculture college in the University of Baghdad, Iraq. The buds were rinsed with deionized water and left to dry in the dark away from direct sunshine for 15 days. The dried leaves were ground into a fine powder using an electronic grinder. A total of 100 g of the fine powder of buds of Syzgium aromaticum was subjected to Soxhlet extraction utilizing distilled water for aqueous extraction for 72 h. Then, the resulting solution was filtered using Whatman No.1 filter paper (Whatman, USA) to eliminate any unwanted particles, and then the extract was concentrated using a rotary flash evaporator at 45°C. The extract was maintained at room temperature in air-tight vials for subsequent investigations as per previously established standards [13].

Ag, Cu, and Ag–Cu NPs were produced under controlled and standardized conditions, following established methods with minor modifications [14,15]. In a 250 mL flask, 10 mL of Syzygium aromaticum extract (100 mg·mL⁻¹) was mixed with 90 mL of a freshly prepared silver nitrate solution (AgNO₃, AFCO, Jordan). A separate flask contained the same volume of plant extract combined with copper nitrate (Cu(NO₃)₂, AFCO, Jordan). The mixtures were continuously stirred at 200 rpm using a magnetic stirrer and then left at room temperature in a dark setting. After 24 h, the solutions became turbid and reddish-brown, indicating the successful synthesis of AgNPs, while the copper solution transitioned from light brown to dark brown. Following centrifugation at 15,000 rpm for 20 min to separate the Syzygium aromaticum extract, the resulting precipitate was washed three times with sterilized and deionized water to remove the unwanted residues. The final precipitate was dried to yield powdered Cu and AgNPs. For the production of bimetallic Ag–Cu NPs, a straightforward one-pot biosynthetic approach was used. Forty-five milliliters of each metal salt (AgNO₃ and Cu(NO₃)₂) were combined in a 250 mL beaker and stirred continuously at room temperature. Subsequently, 10 mL of plant extract was added to this mixture and incubated overnight in the dark. An observable color change in the solution confirmed the formation of Ag–Cu bimetallic NPs. The same washing and drying techniques applied to the monometallic NPs were used to obtain the powdered Ag–Cu NPs [16,17] (Figure 1).

Figure 1

Procedure of producing mono- and bimetallic green NPs from Syzgium aromaticum.

2.2 Spectroscopic characterization of Ag and Cu NPs

The solution absorbance was measured using a UV-Vis spectrophotometer to identify the produced NPs, with wavelengths ranging from 200 to 800 nm. A 1 mL aliquot of the silver NP solution was combined with purified water to achieve a total volume of 9 mL. The infrared analysis was also applied to detect the functional group of the samples, and XRD was employed to investigate the NP structures. Thin films of AgNPs, CuNPs, and Cu-Ag were prepared by depositing 1 mL of each solution onto 1 cm² glass slides, which were then dried by heating on a hot plate. The grain size of the NPs was determined using the Scherrer equation: D = KλβcosθD = KD = Kβcos, where D is the crystallite size, K is the shape factor, λ is the wavelength of the X-rays, β is the line broadening at half-maximum intensity (FWHM), and θ is the Bragg angle. The system utilized Cu-Kα radiation with a wavelength of λ = 1.5406 A° generated at 40 kV. The samples were scanned over a range of 10–70 at room temperature [15]. Furthermore, zeta potential analysis was performed to investigate the stability of the prepared NPs.

2.3 Morphological characterization

The morphology of the NPs was studied using TEM (Tokyo, Japan). The sizes and diameters of the NPs were measured among approximately 25 particles for each prepared nanosolution. The median diameter of the NPs was determined to be over 25 NPs for each sample using TEM images, Image J software, and Excel, and the findings are shown as mean ± SD.

2.4 Antibacterial activity

The Laboratory of Bacteriology at the University of Technology’s College of Applied Science supplied both Gram-positive (S. saprophyticus) and Gram-negative (E. coli) bacterial strains. Mueller–Hinton agar medium (Mast, England) was prepared, the microbiological suspension (E. coli and S. saprophyticus) was set up in comparison with the standard McFarland tube, and 10⁶ CFU·mL⁻¹ (0.5 McFarland standards) was used to assess the antibacterial impact. Using a sterile well cutter, the plates were swabbed and then punched into the agar. Different quantities of NP solution (12.5, 25, 50, and 100 μg·mL⁻¹) were selected based on literature-reported effective doses for similar NPs. This range ensured detectable inhibition zones while minimizing nonspecific toxicity, with sterile deionized water serving as the controlling factor. For the whole experiment, the dishes were incubated at 37°C. The inhibition zone was measured and monitored after incubation [18,19].

2.5 Antibiofilm and bacterial ROS production assay

The bacterial culture grown overnight was diluted to a final concentration of 1 × 10⁶ CFU·mL⁻¹ for the biofilm inhibition experiment. A 96-well plate was filled with 200 µL of this solution, and it was incubated for 5 h at 37°C. Following the incubation time, the solution was incubated for a further 19 h at 37°C with various doses of AgNPs, CuNPs, and their combined form, Ag–Cu NPs (12.5, 25, 50, and 100 μg·mL⁻¹). Once the static biofilm had developed on the 96-well plate walls for 24 h, the medium was gently removed, and the biofilm was washed twice with sterile water. After 20 min of 0.1% crystal violet staining, a water wash, and a 1-h room temperature drying period, the biofilms were left to dry. To remove the stain, 200 µL of 100% ethanol was added to the biofilms that had been stained, and the samples were swiftly agitated for 15 min. Using an ELISA plate reader, the absorbance of extracted crystal violet was determined at 590 nm [20]. In contrast, bacterial strains at a concentration of 1 × 10⁸ CFU·mL⁻¹ were treated with 100 µg·mL⁻¹ AgNPs, CuNPs, and Ag–Cu NPs for 2 h to evaluate ROS generation. H2DCF-DA was used to identify ROS. After cleaning with PBS, bacterial samples were suspended in 1.8 mL of PBS. A total of 200 μL of 1 mM H2DCF-DA was then added to the samples, and they were left in the dark at 37°C for 25 min. Following their collection, the bacterial samples were washed with PBS. The bacterial suspension was allowed to air-dry naturally at room temperature under dark conditions. Subsequently, ROS production was assessed using fluorescence microscopy [21].

2.6 Antibiotic susceptibility and synergistic effect

Using the Kirby–Bauer disk diffusion method, the antibiotic sensitivity of bacterial isolates was evaluated. The microbial suspension was made and compared with the conventional McFarland tube using Mueller–Hinton agar medium; the McFarland standard was used to obtain suspensions at 1.5 × 10⁶ CFU·mL⁻¹ (0.5 McFarland standard). The bacterial suspension (0.2 mL) was spread over the surface of the culture medium evenly using cotton swabs. The dishes were allowed to dry at room temperature. Then, the antibiotic disks were placed with sterile forceps, and the dishes were incubated at 37°C for 24 h. Replicates were made for each dish. The tested antibiotics for each of E. coli included gentamicin (CN-10), augmentin (AMC), ceftriaxone (CTR-5), and trimethoprim/sulfamethoxazole (SXT) antibiotics, while for Sp. Saprophyticus, tetracycline (TE-30), AMC, amoxicillin (AM), and aeftriaxone (CTR-5) antibiotics were used [22]. Using the agar well diffusion assay, the antibacterial potential of the produced samples (AgNPs, CuNPs, and Ag–Cu NPs) was examined against Gram-positive and Gram-negative bacteria [23]. To determine the synergistic action against the previously mentioned bacteria, fold increase (FI,%) = (b − a)/a × 100 was determined, where a and b represent the antibiotic’s zone of inhibition alone and with Ag, Cu, and Ag–Cu NPs, respectively [24,25].

2.7 In silico analysis

The in silico analysis was performed to detect the antibacterial activities of the AgNPs, CuNPs, and Ag–Cu nanocomposites. Materials Studio software was used to establish the 3D crystals of the following NPs. Two types of bacteria were chosen to examine the bioactivities of the compounds (MltA from E. coli, ID: 2GAE, and S. saprophyticus, ID: 3Q6A). The PDB files of the targeted receptors were obtained from www.rcsb.org. Many preparations were taken, starting by removing all the hetatoms from the receptors using the Biovia Discovery Studio program [26]. Then, the missing parts were checked, and the total energy of the proteins was minimized using the SWISS-PDBVIEWER (version 4.1.0) (SPDBV) program [27]. The ligands were saved in the PDBQT format using Autodock 4.2, facilitating the molecular docking process. A grid box was configured to cover the entire surface of the selected proteins, and the docking parameters were optimized using genetic algorithms. Hydrophobic interactions between the docked drug-like molecules and the target proteins were identified using LigPlot + (v.2.2.8). Biovia Discovery Studio was employed to visualize the best-fitting conformers [28].

2.8 Statistical analysis

Statistical analysis was performed using the Graph Prism tool to assess the significance of the antimicrobial, antibiofilm, and synergistic effects of silver (Ag), copper (Cu), and bimetallic Ag–Cu NPs. All experiments were conducted in triplicate to ensure the accuracy and reliability of the results, and the data are expressed as mean ± SD [29–31].

3 Results and discussion

The aqueous solution of Syzgium aromaticum extract was used in this work as a physiologically active green reducer for reducing copper and silver ions to Ag, Cu, and bimetallic Ag–Cu NPs owing to the phytochemical components present in the plant extracts. The progress of the reaction was monitored using spectrophotometric analysis.

3.1 Bacterial isolation and identification

The strongest and most productive isolates for the biofilm were selected. To identify the bacterial isolates of S. saprophyticus and E. coli, the isolates were analyzed using the VITEK 2 system, which is an identification method that relies on biochemical interactions between the media in the VITEK-2 identification cards (GP, GN); the bacterial isolates were suspended in their solutions to identify the isolates using the identification card for each type of microorganism.

3.2 UV-Vis spectrophotometry analysis

The UV–Vis (ultraviolet–visible) spectrometric absorbance of the generated AgNPs, CuNPs, and Ag–Cu NPs was determined in the range of 200–800 nm. The absorption maxima, λ _max, at 382 and 564 nm in Figure 2 show that the liberated electrons in the metal NPs generated the surface plasmon resonance (SPR) absorption band for the Ag and CuNPs, respectively, and a broad shifted peak was also observed in the range of 399–526 nm with the peak at 487 nm for the Ag–Cu nanocomposite, which is a strong indicator for the bimetallic NP formation. All peaks correspond to the free electrons of the metals at the nanoscale size, which results in SPR. This is consistent with both, and it is created when the metal’s electrons oscillate in response to light waves [32,33].

Figure 2

UV-Vis analysis of the Ag, Cu, and Ag–Cu bimetallic NPs. The peaks correspond to the free electrons of the metals at the nanoscale size that result in SPR. Ag NPs (382 nm), CuNPs (564 nm), and Ag–Cu nanocomposite (487 nm).

3.3 Fourier-transform infrared (FTIR)spectroscopy analysis

The samples were characterized using an FT-IR device (Shimadzu, Kyoto, Japan) and KBr series. As shown in Figure 3, the FT-IR spectra of Ag, Cu, and Ag–Cu NPs display many bands indicative of the stretching and bending vibrations of the O–H, C–C, and C–O functional groups. The strong vibrations between 3,000 and 4,000 cm⁻¹ are linked to the OH groups, strong vibrations between 1,500 and 2,000 cm⁻¹ are linked to the carbonyl groups of the acid group, and strong vibrations between ∼1,500 and 2,000 cm⁻¹ are linked to amide groups, based on the images captured by the FT-IR device. The presence of functional groups associated with both metals, along with additional minor peaks, confirms the synthesis of bimetallic NPs. The Ag–Cu FT-IR images are also a little bit flatter [34,35].

Figure 3

FTIR analysis of the Ag, Cu, and Ag–Cu bimetallic NPs.

3.4 XRD analysis

When comparing XRD results with JCPDS File no. 04-0783, in Figure 4, four Ag NP-related peaks can be seen at angles 2θ (38.1°, 47.08°, 67. 9°, and 78.9°), which correspond to the (111), (200), (220), and (311) planes and two peaks for Cu at angles for 2θ (43.04° and 51.3°) corresponding to the (111*) and (200*) planes, respectively. Using Scherrer’s equation, the crystallite size of 32 nm is shown by the sharpest peak (111), which matches the particle size distribution calculated according to the TEM images [36].

Figure 4

XRD analysis of (a) AgNPs, (b) CuNPs, and (c) Ag–Cu bimetallic NPs.

3.5 Zeta potential analysis

The zeta potential was used to test the stability and surface charge of the prepared NPs and their conjugates. Highly stable colloids presented zeta potentials, as shown in Figure 5.

Figure 5

Zeta potentials for AgNPs (a), CuNPs (b), and Ag–Cu NPs (c).

3.6 Morphological examination of NPs using TEM

AgNPs, CuNPs, and Ag–Cu NPs were analyzed using TEM to reveal their morphological characteristics and structural details. These included spherical shapes, some accumulation and aggregation of the NPs, and an average mean size of 28.02 nm, as measured using ImageJ software, as shown in Figure 6: AgNPs (right panel), CuNPs (left panel), and Ag–Cu NPs (middle) [37,38].

Figure 6

TEM images and size distribution of AgNPs (right panel; scale bar: 75 nm), Ag–Cu NPs (middle panel; scale bar: 100 nm), and CuNPs (left panel; scale bar: 50 nm).

3.7 Antibacterial activity of the prepared NPs

Table 1 shows the results of utilizing the agar well-diffusion method to determine the antibacterial activity of AgNPs, CuNPs, and Ag–Cu NPs against Gram-negative bacterial strains (E. coli) and Gram-positive bacteria (S. saprophyticus) at different concentrations (12.5, 25, 50, 100 μg·mL⁻¹). The results show that AgNPs and CuNPs have a slight effect on E. coli and S. saprophyticus.

Table 1

Antibacterial activities of CuNPs, AgNPs, and Ag–Cu bimetallic NPs against DF isolates

Microorganism	Inhibition zone (mm) at different concentrations (μg·mL⁻¹)
Microorganism	12.5	25	50	100
E. coli	Ag NPs
	16.93 ± 0.29	17.40 ± 0.69	19.6 ± 0.39	14.93 ± 0.42
	CuNPs
	13.40 ± 0.23	14.73 ± 0.68	15.79 ± 0.69	15.93 ± 0.24
	Ag–Cu NPs
	13.37 ± 0.34	20.23 ± 0.73	26.73 ± 0.55	27.33 ± 1.01
S. saprophyticus	AgNPs
	13.80 ± 0.76	14.13 ± 065	15.33 ± 0.67	16.70 ± 0.26
	CuNPs
	10.63 ± 0.27	15.30 ± 0.51	17.30 ± 0.66	20.07 ± 0.36
	Ag–Cu NPs
	15.90 ± 1.03	20.83 ± 1.15	26.77 ± 0.85	29.53 ± 0.45

The synthesized Ag–Cu NPs had the greatest effect on the growth of S. saprophyticus bacteria, with an average diameter of the inhibition zone of 29.53 ± 0.45 mm at a 100 μg·mL⁻¹ concentration, followed by E. coli with an average diameter of the inhibitory zone of 27.33 ± 1.01 mm. In comparison with AgNPs and CuNPs, at a 100 μg mL⁻¹ concentration, the inhibitory zone was 20.07 ± 0.36 and 16.70 ± 0.26 mm against S. saprophyticus while 15.93 ± 0.24 and 14.93 ± 0.42 mm against E. coli, as illustrated in Figure 7. According to a previous study, Gram-positive S. saprophyticus cells exhibit a high concentration of carboxyl and amine functional chains on their surface, which are highly attracted to copper ions [39]. By creating a cross-link between the nucleic acid strands, the interaction of copper ions with the bacterial DNA results in abnormalities in the helical structure. This impedes the body’s metabolism in several ways, impairing Gram-negative bacteria’s ability to function as enzymes and causing cell death. The potential of copper and silver ions to infiltrate bacterial cells, particularly those of E. coli, may be the primary source of the bimetallic copper–silver NP system’s antibacterial activity. Additionally, silver ions accumulate within the cell wall, triggering the release of lipopolysaccharides (LPS) and membrane proteins, further accelerating bacterial death [40].

Figure 7

Antibacterial activity of AgNPs (upper panel), CuNPs (middle panel), and Ag–Cu bimetallic NPs (lower panel) against E. coli (right panel) and S. saprophyticus (left panel) isolates where A is 12.5, B is 25, C is 50, D is 75 and E is 100 μg/ml.

The antibacterial activity is higher against Gram-negative bacteria than against Gram-positive bacteria due to the ability of Gram-positive bacteria’s cell walls to bind larger amounts of metal ions. Furthermore, the molecular composition of their cell walls varies: Gram-positive bacteria have a thick and rigid peptidoglycan layer, whereas Gram-negative bacteria have a thin LPS layer that envelops their peptidoglycan layer, impeding the access of NPs to the cell walls [41,42].

It is important to note that bimetallic Ag–Cu NPs synthesized using Syzygium aromaticum extract exhibit strong antibacterial and antibiofilm activity against E. coli and S. saprophyticus. The results of NPs produced via other methods (chemical, physical, or fungal-mediated synthesis) are compared.

4 Synergistic effects of NPs and antibiotics

This test was carried out to determine susceptibility to various harmful microorganisms. The combined AgNPs, CuNPs, Ag–Cu NPs, and antibiotics were also subjected to the susceptibility test. Table 2 provides a summary of the findings.

Table 2

Synergistic effect of monometallic and bimetallic NPs against different bacterial strains by measuring the diameter of the inhibition zones (mm) and the percentage of fold increase

Bacteria	A name	Inhibition zone (mm)
Bacteria	A name	A	A + AgNPs	FI%	A + CuNPs	FI%	A + Ag–Cu NPs	FI%
E. coli	CTR	12.8	11.2	−13.85	14.3	10	17.6	37.5
	AMC	10	10.3	3	8.6	−14	13.5	35
	SXT	6	6.3	5	6	0	12.4	106.67
	CN	25.3	25.1	−0.79	29.2	15.42	18.9	−25.3
S. saprophyticus	CTR	15	14.9	−0.67	15.8	5.33	15.8	5.34
	AMC	6	10	66.67	8.9	48.33	6	0
	AM	6	8.7	45	7.1	18.33	10.1	68.34
	TE	25.7	29.1	13.23	30.1	17.12	26.3	2.34

Note: A: antibiotic; FI: fold increase; the diameter of the antibiotic disk is 6 mm.

As shown in Table 2, E. coli is generally resistant to antibiotics such as AM and its combination, and most isolates begin to develop resistance against trimethoprim. In addition, E. coli demonstrates intermediate resistance to ceftriaxone (CTR), and it is strongly sensitive to gentamicin (CN). However, Gram-positive bacteria S. saprophyticus demonstrated a high level of resistance to AM, followed by its combination from AMC (AM/clavulanate), and also demonstrated sensitivity to tetracycline (TE) according to the EUCAST standardized disk diffusion method [43–45]. Four different antibiotics were used for each bacterial isolate, and all types of NPs were used according to their minimum inhibitory concentration. The effect of bimetallic NPs and its synergistic effect with the selected antibiotics showed that most Ag–Cu NP combinations (e.g., +37.5% with CTR, +106% with SXT) show significant enhancement and increased diameter of the inhibition zone of trimethoprim/SXT when combined with the Ag–Cu composite and slight increase with CTR. However, it showed an antagonism effect on CN with no noticeable effect on AMC (AM/clavulanate); these antagonistic effects in selected cases (e.g., with CN) likely arise from competitive binding or metal–antibiotic incompatibility that can degrade certain antibiotics reducing efficacy, but the overall trend strongly supports synergistic enhancement. The mono NPs showed different results: CuNPs showed a significant increase in the effect of CN. All results related to E. coli are shown in Figure 8 [46,47]. The other four antibiotics were tested against S. saprophyticus: the bimetallic NPs increased the AM effect, while no relevant effects were observed on the other antibiotics. On the other hand, AgNPs showed an increase in the efficacy of augmentin (AUG) and TE. Furthermore, CuNPs increased AUG and TE significantly, as shown in Figure 8. The combination of antibiotic and AgNPs, CuNPs, and Ag–Cu bimetallic NPs increased the inhibition zone for most antibiotics, varying in effectiveness for each type of NP. Some others revealed a backward action when combined with some antibiotics. Overall, Ag–Cu NPs alone outperform monometallic NPs (Table 1), and most antibiotic combinations improved the synergistic effects of the investigated compounds.

Figure 8

Antibiotic susceptibility test to determine the synergistic relationship between antibiotic and NPs against (a) E. coli and (b) S. saprophyticus.

This study demonstrates how the antibiotic and mono- and bimetallic NP combination increased the antibacterial activity against microorganisms. Recent studies generally proposed that Ag affects bacterial plasma membranes, whereas Cu breaks down nucleic acids and other internal macromolecules and cells. Also, it has been claimed that Cu ions produced from CuO alloys inactivate bacteria by enhancing ROS generation causing DNA damage and lipid peroxidation [48,49], and the coupling of the antibiotic and NPs could inhabit the DNA gyrase enzyme and topoisomerase IV activities that limits the bacterial cells’ ability to divide, ultimately leading to cell death [48]. The amount of drug needed when the NPs and antibiotics are combined is minimal, thereby reducing the drug’s toxicity and the chance of bacterial resistance.

5 Inducing bacterial ROS generation by NPs

The ability of Ag, Cu, and Ag–Cu NPs to induce ROS production in bacterial cells was evaluated using H₂DCF-DA staining and fluorescence microscopy. Figure 9 demonstrates that untreated bacterial cells exhibited minimal fluorescence, indicating low baseline ROS levels. In contrast, treatment with NPs resulted in significantly increased fluorescence intensity, demonstrating ROS accumulation. Among the tested NPs, Ag–Cu bimetallic NPs showed the most pronounced effect, generating substantially higher ROS levels compared to their monometallic counterparts. At a concentration of 50 µg·mL⁻¹, Ag–Cu NPs already induced strong fluorescence, while Ag and Cu NPs required higher concentrations (75–100 µg·mL⁻¹) to achieve similar effects [49,50]. An inner oxidative stress test was performed using DCFH-DA, a non-fluorescent, cell-permeable precursor of DCF, which is a useful tool for monitoring the redox state of the sample for variations in fluorescence. Bacterial cells treated with monometallic NPs show relatively little influence on viability when compared to the control group; however, cells exposed to Ag–Cu NPs display a greatly enhanced signal. Additionally, compared to E. coli, S. saprophyticus produces a comparatively higher amount of fluorescence. The robust ROS production induced by Ag–Cu NPs likely contributes to their antimicrobial efficacy through multiple mechanisms. The simultaneous release of Ag⁺ and Cu²⁺ ions from bimetallic NPs may create a synergistic oxidative stress response, overwhelming bacterial antioxidant defenses. This oxidative damage to cellular components, combined with direct membrane disruption, provides a comprehensive antibacterial action that explains the enhanced performance of Ag–Cu NPs observed throughout this study. This test result indicates that direct contact with NPs causes bacterial cells to be significantly impacted by the formation of free radicals [51,52].

Figure 9

ROS generation for both S. saprophyticus and E. coli in the presence of Ag, Cu, and Ag–Cu NPs. Lower panel: Fluorescence intensity of generated ROS.

6 Antibiofilm activity of mono- and bimetallic NPs

By assessing biofilm formation with crystal violet in the presence of different concentrations (12.5, 25, 50, 100 μg·mL⁻¹) of each type of NP employed in this work, the antibiofilm activity of Ag, Cu, and Ag–Cu NPs was assessed. At 5 × 10⁷ CFU·mL⁻¹, there was a significant difference in the percentage of biofilm biomass of S. saprophyticus and E. coli when compared to the control group (*p < 0.05, **p < 0.01). The biofilms at the bottom of the 96-well plates were marginally eliminated by the Ag, Cu, and Ag–Cu NP treatments, as illustrated in Figures 10 and 11. However, the experimental group that received Ag, Cu, and Ag–Cu NP treatments showed considerable biofilm removal. According to these findings, of all the microorganisms examined, the Ag–Cu nanocomposite had the highest anti-biofilm impact when compared to other groups. Figure 9 shows the biofilm quantification against E. coli using a semi-quantitative study of crystal violet staining. In comparison to AgNPs and CuNPs, the Ag–Cu nanocomposite demonstrated better anti-biofilm action against both Gram-positive and Gram-negative bacteria. There was a substantial difference in the biofilm destruction of E. coli and the Ag–Cu nanocomposite when compared to the control group (*p < 0.05, **p < 0.01). The outcome further showed that the nanocomposite’s anti-biofilm effectiveness was greatly increased by the complementary antibacterial action of Ag and CuNPs, resulting in the breakdown of the biofilms.

Figure 10

Effect of different concentrations of Ag-NPs, Cu-NPs and Ag-Cu nanocomposite on the initial attachment and biofilm formation by E. coli.

Figure 11

Effect of different concentrations of Ag-NPs, Cu-NPs, and Ag–Cu nanocomposite on the initial attachment and biofilm formation by S. saprophyticus.

Bacterial cells must synthesize and secrete exopolysaccharides (EPSs) for biofilms to develop, resulting in the formation of bacterial biofilms. The production of EPS is triggered by environmental stimuli, which the bacteria react to. Therefore, biofilm development will be limited if it is possible to block or prevent the synthesis of EPS. Our experiment on AgNPs’ anti-biofilm activity was built around this idea [53]. Ag, Cu, and Ag–Cu bimetallic NPs exhibit potent antibacterial and antibiofilm properties through several mechanisms. Ag NPs disrupt bacterial cell membranes, generate ROS, and release silver ions that interfere with protein functions. CuNPs similarly induce oxidative stress, release copper ions, and cause membrane damage. When combined into Ag–Cu bimetallic NPs, these effects are synergistically enhanced, leading to increased ROS production and dual metal ion release, which disrupt multiple bacterial processes simultaneously. This combination is particularly effective against biofilms, protective layers formed by bacterial communities, making Ag–Cu bimetallic NPs a powerful tool in combating persistent bacterial infections [54,55].

6.1 Molecular docking simulation

Despite the solid antibacterial bioactivity of silver and copper elements, the inhibitory effect of the NP compounds was fragile. The docking interaction resulted in a positive free energy of binding, indicating a lack of inhibitory action. The composition of the crystals, whether containing only silver, copper, or both, limited the interactions to only the metal–acceptor interactions or the metal–donor repulsions [56]. There was no H-bonding or electrostatic attractions. Also, the spherical crystals would prefer interactions with the edges of the protein rather than being immersed in the protein body, which is preferable thermodynamically. Ag NPs exhibited the strongest complexation with both the 2GAE and 3Q6A receptors, followed by CuNPs, and the Ag/Cu nanocomposite showed the weakest interaction. For 2GAE, Ag NPs recorded the lowest binding free energy at +0.3 kcal·mol⁻¹, and 3Q6A displayed a value of 0.39 kcal·mol⁻¹. Figures 12 and 13 illustrate the interaction positions of the best conformations of Ag NPs, CuNPs, and Ag/Cu nanocomposites while docking with 2GAE and 3Q6A [57]. In Figures 11 and 14, AgNPs and CuNPs occupied comparable positions around 2GAE. At the same time, the Ag/Cu nanocomposite exhibited a different, though nearby, position, as specified by the coordinate attributes in Table 3. Figures 13 and 15 show the interactions and the hydrophobicity of the nanocompounds with different amino acids of the studied proteins. By concentrating on the interactions of Ag NPs, it interacted with chain A of 2GAE through HIS238, GLY235, GLU240, and ASN171. Ag NPs interacted with chain G of 3Q6A through GLN108 and GLU94, as shown in Table 3 [58]. Note that docking only assesses one aspect (protein binding), while experimental results reflect the net antibacterial effect, which includes ROS generation, embrane disruption, synergistic ion toxicity, and biofilm penetration. Ag–Cu NPs likely exploit multiple pathways that are not fully captured by static docking simulations. However, the Ag–Cu NPs also showed a good binding capability and molecular docking only predicts protein binding affinity, whereas experimental antibacterial activity depends on multiple synergistic mechanisms and antibacterial activity depends on broader cellular damage (Tables 4 and 5).

Figure 12

The positions of the best conformations of AgNPs, CuNPs, and Ag/Cu nanocomposite around 2GAE receptors.

Figure 13

3D image and the hydrophobic interactions of (a) AgNPs, (b) CuNPs, and (c) Ag/Cu nanocomposite with 2GAE receptor.

Figure 14

The positions of the best conformations of AgNPs, CuNPs, and Ag/Cu nanocomposite around the 3Q6A receptor.

Table 3

Comparison between the two targeted proteins interacting with AgNPs, CuNPs, and Ag/Cu nanocomposite through binding energies, Ref. RMSD, and possible interactions

Parameter	AgNPs	CuNPs	Ag/Cu nanocomposite
2GAE receptor
Free energy of binding (kcal·mol⁻¹)	+0.3	+1.09	+2.19
Ref. RMSD	27.91	28.29	43.79
Attributes of the best conformer	X = 8.114	X = 7.788	X = 2.671
	Y = 28.504	Y = 28.524	Y = 43.866
	Z = −6.265	Z = −6.849	Z = −17.833
Metal–acceptor interactions	LIG Ag: HIS238(A) O,	LIG Cu: ASN171(A) O.	LIG Ag: TYR208(A) O.
	2.97 Ao, 3.08 Ao.	LIG Cu: GLY235(A) O.	LIG Ag: ARG209(A) O.
	LIG Ag: GLY235(A) O.	LIG Cu: HIS238(A) O,	LIG Ag: ASN250(A) O.
	LIG Ag: GLU240(A) O.	2.85 Ao, 3.37 Ao.	LIG Cu: VAL181 (A) O.
	LIG Ag: ASN171(A) O.	LIG Cu: GLU240(A) O,	LIG Cu: ASP180(A) O.
		2.88 Ao, 2.97 Ao.
3Q6A receptor
Free energy of binding (kcal·mol⁻¹)	+0.39	+1.16	+2.08
Ref. RMSD	99.25	101.06	99.17
Attributes of the best conformer	X = −5.661	X = −5.832	X = −5.796
	Y = 42.240	Y = 42.540	Y = 47.310
	Z = 92.565	Z = 94.080	Z = −87.796
Metal–acceptor interactions	LIG Ag: GLN108(G) O.	LIG Cu: GLU94(G) O.	LIG Ag: GLY111(G) O.
	3.26 Ao, 3.29 Ao, 3.31 Ao.	LIG Cu: TYR48(G) O,	LIG Cu: GLU50(G) O.
	LIG Ag: GLU94(G) O.	2.89 Ao, 3.27 Ao.

Figure 15

3D image and the hydrophobic interactions of (a) AgNPs, (b) CuNPs, and (c) Ag/Cu nanocomposite with 3Q6A receptor.

Table 4

Conformations detected from the blind docking simulation between AgNPs, CuNPs, and Ag/Cu NPs and 2GAE receptors

Conformer’s no.	Binding energy of AgNPs with 2GAE receptor (kcal·mol⁻¹)	Binding energy of CuNPs with 2GAE receptor (kcal·mol⁻¹)	Binding energy of AgCu nanocomposite with 2GAE receptor (kcal·mol⁻¹)
1	+0.93	+1.47	+2.75
2	+0.50	+1.48	+3.01
3	+1.02	+1.46	+2.73
4	+1.03	+1.58	+2.62
5	+0.55	+1.60	+2.19
6	+0.99	+1.47	+2.60
7	+0.31	+1.49	+3.21
8	+0.55	+1.48	+2.67
9	+0.77	+1.26	+2.76
10	+0.84	+1.26	+2.54
11	+0.89	+1.44	+2.77
12	+0.30	+1.44	+2.83
13	+0.88	+1.47	+2.76
14	+0.32	+1.49	+2.69
15	+0.81	+1.10	+2.85
16	+0.31	+1.48	+3.06
17	+0.51	+1.48	+2.44
18	+1.15	+1.35	+2.83
19	+0.86	+1.10	+2.58
20	+0.95	+1.44	+2.21
21	+ 1.04	+1.26	+2.65
22	+0.80	+1.36	+2.83
23	+1.06	+1.48	+2.53
24	+0.94	+1.11	+2.90
25	+0.94	+1.46	+2.82
26	+0.80	+1.33	+2.89
27	+0.86	+1.53	+2.70
28	+0.80	+1.10	+2.71
29	+0.85	+1.50	+2.50
30	+0.78	+1.34	+3.07
31	+1.15	+1.09	+2.53
32	+0.86	+1.28	+2.57
33	+1.05	+1.49	+3.00
34	+0.73	+1.35	+2.63
35	+0.53	+1.27	+2.84
36	+0.84	+1.09	+2.97
37	+0.94	+1.12	+2.76
38	+0.51	+1.41	+2.68
39	+0.91	+1.41	+2.66
40	+0.80	+1.09	+3.19
41	+0.85	+1.09	+2.74
42	+0.87	+1.09	+2.82
43	+0.78	+1.10	+2.62
44	+0.81	+1.09	+2.70
45	+0.79	+1.51	+2.47
46	+0.88	+1.47	+2.44
47	+1.13	+1.35	+2.62
48	+0.30	+1.49	+2.93
49	+1.05	+1.49	+2.44
50	+0.88	+1.46	+2.39

Table 5

Conformations detected from the blind docking simulation between AgNPs, CuNPs, and Ag/Cu NPs and the 3Q6A receptor

Conformer’s no.	Binding energy of AgNPs with 3Q6A receptor (kcal·mol⁻¹)	Binding energy of CuNPs with 3Q6A receptor (kcal·mol⁻¹)	Binding energy of the AgCu nanocomposite with 3Q6A receptor (kcal·mol⁻¹)
1	+1.00	+1.25	+2.74
2	+0.83	+1.24	+2.38
3	+0.75	+1.45	+2.72
4	+0.97	+1.48	+2.72
5	+0.80	+1.47	+2.86
6	+0.39	+1.52	+2.92
7	+0.42	+1.57	+2.83
8	+0.93	+1.55	+3.02
9	+0.85	+1.53	+3.15
10	+1.01	+1.25	+2.93
11	+0.90	+1.34	+2.36
12	+1.04	+1.43	+2.77
13	+0.89	+1.18	+2.51
14	+1.00	+1.61	+2.74
15	+0.62	+1.49	+2.67
16	+0.87	+1.16	+2.29
17	+0.73	+1.24	+2.85
18	+0.75	+1.26	+2.77
19	+0.96	+1.48	+3.02
20	+0.97	+1.61	+3.05
21	+0.96	+1.49	+2.12
22	+0.41	+1.54	+2.92
23	+1.03	+1.48	+2.70
24	+0.56	+1.45	+2.62
25	+0.46	+1.51	+2.94
26	+0.90	+1.41	+2.55
27	+0.70	+1.56	+2.71
28	+0.81	+1.61	+2.64
29	+0.97	+1.19	+2.69
30	+0.67	+1.22	+2.85
31	+0.91	+1.54	+2.88
32	+1.00	+1.50	+2.59
33	+0.84	+1.56	+2.78
34	+0.94	+1.54	+2.08
35	+0.86	+1.61	+2.88
36	+0.93	+1.23	+2.31
37	+1.06	+1.53	+2.46
38	+0.64	+1.50	+2.85
39	+0.87	+1.51	+2.09
40	+0.83	+1.47	+3.04
41	+1.30	+1.47	+2.73
42	+0.85	+1.50	+2.88
43	+0.69	+1.69	+3.28
44	+0.62	+1.51	+2.15
45	+0.83	+1.47	+2.54
46	+1.08	+1.47	+2.60
47	+0.91	+1.47	+2.96
48	+0.97	+1.24	+2.69
49	+0.51	+1.22	+2.95
50	+1.19	+1.61	+3.01

7 Conclusions

This study successfully demonstrated the green synthesis of silver (Ag), copper (Cu), and bimetallic Ag–Cu NPs using Syzygium aromaticum extract as a reducing agent. The bimetallic Ag–Cu NPs exhibited superior antibacterial activity compared to their monometallic counterparts, effectively targeting both E. coli and S. saprophyticus. The enhanced efficacy is attributed to the synergistic effects of silver and copper, which disrupt bacterial membranes, induce oxidative stress, and damage cellular components. Additionally, the bimetallic NPs demonstrated significant antibiofilm activity, an important feature in combating persistent infections caused by biofilm-forming bacteria. Their ability to inhibit biofilm formation and disrupt established biofilms suggests they could be highly effective against challenging bacterial infections, such as UTIs and HAP. Furthermore, the study revealed that combining the NPs with conventional antibiotics enhanced the drugs’ efficacy, reducing the antibiotics needed and potentially mitigating the risk of resistance. The synergistic effect between Ag–Cu NPs and antibiotics like CN and TE underscores their potential in clinical applications. The promising results from this research open new avenues for developing NP-based antimicrobial treatments, especially against drug-resistant and biofilm-associated infections. However, further studies are needed to evaluate the NPs’ safety, pharmacokinetics, and environmental impact to ensure their viability for medical uses.

Acknowledgments:

The authors appreciates University of Technology- Iraq for their support. Also, the authors extend their appreciation to the Researchers Supporting Project number (RSPD2025R971), King Saud University, Riyadh, Saudi Arabia, for funding this research.

Funding information: The authors extend their appreciation to the researchers supporting project number (RSPD2025R971) at King Saud University, Riyadh, Saudi Arabia, for funding this study. They also appreciate the University of Technology, Iraq, for the logistical support of this work.
Author contributions: Jafar Abdulkareem Abbas, Buthenia Abd Alhamza Hasoon, and Majid Sakhi Jabir: writing original draft, methodology, investigation, and formal analysis. Buthenia Abd Alhamza Hasoon and Majid Sakhi Jabir: main concept, data interpretation, and supervision. Majid Sakhi Jabir, Buthenia Abd Alhamza Hasoon, Suresh Ghotekar, and Ayman Abdel-Aziz Swelum: writing – review and editing, visualization, and data curation. All authors reviewed the manuscript.
Conflict of interest: The authors state no conflict of interest.
Data availability statement: All data generated or analyzed during this study are included in this published article.

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Received: 2025-01-09

Accepted: 2025-05-12

Published Online: 2025-07-23

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/gps-2025-0009

Keywords for this article

Ag-Cu NPs; antibacterial; antibiofilm; MDR; ROS; in silico study

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