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Modulation of Staphylococcus aureus antibacterial activity based on silver concentration in ZnO/Ag system
*Corresponding author: E-mail address: ventura.rl65@gmail.com (V. Rodriguez-Lugo)
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Received: ,
Accepted: ,
Abstract
The use and development of green synthesis methods have emerged as a sustainable strategy for producing nanomaterials. These involve using plant extracts as natural reducing and stabilizing agents. In this study, an antibacterial investigation was conducted on zinc oxide (ZnO) and zinc oxide/silver (ZnO/Ag) nanostructures at various silver (Ag) concentrations (0.5, 1, 2, 3, and 8 wt%), synthesized using an infusion of Origanum vulgare combined with the solid-state method at 500°C. The synthesized products were characterized using X-ray diffraction (XRD), identifying the hexagonal wurtzite phase (ZnO) with a preferential orientation on the [101] direction and a crystallite size of 30.63 nm, and the corresponding Ag phase with a preferential orientation in [111] direction, which exhibited an increase in crystallite size from 29.66 to 72.26 nm depending on the Ag concentration. Fourier transform infrared spectroscopy (FTIR) confirmed the presence of functional groups from the extract in the initial samples and their elimination after thermal treatment. Transmission electron microscopy (TEM) analysis showed that the synthesized nanoparticles (NPs) are mostly spherical and semi-spherical, with uniform sizes ranging from 19 to 26 nm, depending on the Ag concentration. High-resolution TEM (HRTEM) and fast fourier transform (FFT) allowed for the identification of interplanar distances and crystal planes, confirming the crystalline nature and growth direction of the samples. Finally, the nanomaterials were subjected to antibacterial tests against Staphylococcus aureus and Escherichia coli. The results showed that the samples containing 2% and 3 wt% Ag exhibited the optimal antibacterial activity against S. aureus. In contrast, the inhibition observed against E. coli was lower and more variable, suggesting that the antibacterial performance is bacterium-dependent, likely due to structural differences in their cell walls.
Keywords
Green synthesis
Nanoparticles
Origanum vulgare
Silver
Zinc oxide
1. Introduction
In recent years, research on nanomaterials with antibacterial properties has gained importance as a response to bacterial resistance caused by the excessive use of antibiotics [1,2]. For this reason, the development of new nanomaterials represents a promising alternative to inhibit bacterial infections [3]. In particular, composite materials [4] based on ZnO and ZnO/Ag are noted for their potential to provide bactericidal effects economically and efficiently [5,6]. Moreover, they exhibit a broad spectrum of action against Gram-positive (G+) and Gram-negative (G-) bacteria, making them ideal candidates for antibacterial applications [6].
Although traditional nanoparticles (NPs) such as those made of Cu [7], Ag [8], and Au [9] are known for their biocidal properties, their use is limited by factors such as the high cost of precious metals or their high demand in other sectors. This underscores the need to explore more accessible and efficient alternatives, such as ZnO and ZnO/Ag [10] composite materials [11,12]. One of the main challenges in NPs, regardless of the synthesis method used, is ensuring that they are produced with dimensions suitable for their biocidal applications. Factors such as concentration [13], composition, temperature [1,14], pH, type of precursors, and synthesis routes play a crucial role, as they determine key physicochemical properties like morphology, particle size [15,16], crystalline phase [17], and crystal size [18,19]
The most common methods for synthesizing ZnO/Ag are hydrothermal and solvothermal techniques [20], chemical precipitation [20], green chemistry approaches, particularly green synthesis [21]. Among these, green synthesis has attracted increasing attention due to its sustainability, low cost, and its alignment with ecological principles. This approach helps to reduce or eliminate the generation of toxic waste and decreases energy consumption, as it generally occurs under mild temperature and pressure conditions. Additionally, it minimizes the use of hazardous chemical reagents, making it a safer and more environmentally friendly option [18]. One of its main advantages is its versatility, as it employs plant extracts rich in bioactive compounds such as flavonoids, tannins, and polyphenols. These compounds not only act as natural reducing agents but can also modify the physicochemical properties of the synthesized NPs [22], enhancing both their functionality and biocompatibility [19]. In addition, green synthesis offers the possibility of scaling up the process more economically, establishing itself as a promising alternative to conventional methods for the production of nanomaterials [23].
An example is the use of Origanum vulgare extract, which is a rich source of bioactive compounds such as phenols, flavonoids, phenolic acids, thymol, carvacrol, terpenoids, and rosmarinic and caffeic acids. These secondary metabolites play a dual role in the green synthesis of NPs: they act as reducing agents by transforming metal ions into their nanostructured form, and as stabilizing agents that prevent agglomeration, favoring the formation of NP with small sizes and well-defined morphologies [24]. In a study on the synthesis of Ag NPs, Origanum vulgare demonstrated significant advantages over other plant extracts. For instance, particle sizes between 15-35 nm have been reported, which are smaller than those obtained with Ocimum vulgare, Cymbopogon citratus, and Azadirachta indica, whose size ranges vary between 20-50 nm [25].
Different researchers have explored environmentally friendly synthesis routes to minimize ecological impact. One example is the work of Rajith et al. [26], who synthesized a ZnO/Ag system using the solution combustion method, employing an extract from Calotropis gigantea leaves and nitrates, followed by calcination at 500°C. They observed NPs between 100-150 nm, which exhibited antibacterial properties by inhibiting the growth of human pathogens, showing inhibition zones against E. coli (9.38 mm) and S. aureus (14.46 mm). In addition, these NPs showed photocatalytic activity. In the same year, Thatikayala et al. [27] synthesized ZnO/Ag NPs using a Tamarindus indica pulp extract with 0.5% wt of Ag. After calcining at 600°C for 2 h of the products, NPs of 50-60 nm for ZnO and 38 nm for ZnO/Ag were obtained; Ag showed an epitaxial growth and an average distribution of 5 nm. When they using a concentration of 2 mg/mL of ZnO/Ag with dissolutions of 50, 100, 150, and 200 µL, in inhibition zones measuring 14 ± 0.72, 15 ± 0.49, 16 ± 0.73, and 19 ± 0.45 mm were recorded, respectively, showing remarkable antibacterial activity against G+ and G- bacteria such as Pseudomonas aeruginosa, E. coli, Bacillus subtilis, and S. aureus were observed, respectively.
Jakinala et al. [28] synthesized a ZnO-Ag nanocomposite using a green method with an extract from Stenotaphrum secundatum grass. The products were calcined at 500°C for 4 h, yielding NPs with average sizes of 38.34 ± 7.78 nm for ZnO, 44.61 ± 4.04 nm for Ag, and 57.14 ± 2.82 nm for ZnO-Ag. Antibacterial tests conducted against S. aureus, E. coli, Klebsiella pneumoniae, and Enterococcus faecalis showed a concentration-dependent decrease in the antibacterial efficacy of the nanocomposite depending on the concentration. Notably, a reduction in the inhibition zone for S. aureus was observed, decreasing from 12.2 mm at a concentration of 10 µg/mL to 9 mm at a concentration of 1.25 µg/mL.
Khanjari et al.[29] synthesized ZnO and ZnO/Ag NPs through green synthesis using an extract from Sophora pachycarpa. The products were calcined at 450°C for 3 h, analyzing both the reaction mechanisms and their antibacterial and anticancer properties. Various ratios of ZnO/AgNO₃ (1:1, 1:0.5, and 1:0.2) were used, yielding crystal sizes of 36.4 nm for ZnO and particle sizes of 50-65 nm for ZnO/Ag. Antibacterial evaluations indicated an optimal response against E. coli at concentrations of 62.5 to 250 µg/mL. For S. aureus and E. faecalis, Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined to be 125 and 250 µg mL-1, respectively.
The present research focuses on synthesizing ZnO/Ag NPs by combining green synthesis and solid-state methods, using an extract from Origanum vulgare as a natural reducing agent. This plant possesses antibacterial, antiseptic, and fungicidal properties [30,31]. The study specifically evaluated the microstructural and morphological effects of the system as the concentration of Ag varied from 0.5% to 8 wt% in a ZnO matrix calcined at 500°C for 4 h. Antibacterial tests were conducted against the human pathogen S. aureus using the disk diffusion method on agar. Additionally, the study detailed how the percentage of Ag influences the microstructural, morphological, and antibacterial properties of the ZnO/Ag system, focusing on its activity against G+ and G- bacteria.
2. Materials and Methods
2.1. Extract preparation
The aqueous extract of Origanum vulgare (commonly known as “Mexican oregano”) was prepared following the methodology described by Munguia et al. [32]. Fresh leaves of O. vulgare were collected in the Real del Monte region, Hidalgo, Mexico, during November and December (Step 1, Figure 1). The collected leaves were dried for 48 h at 40°C and then ground in a porcelain mortar to obtain a fine powder (Step 2-3). Subsequently, 12.5 g of the powdered leaves were mixed with 250 mL of boiling deionized water and stirred at 250 rpm for 30 min (Step 4). A light brown-colored infusion was obtained as a result of this process. The resulting infusion was filtered at room temperature and stored at 4°C until further use (Step 5).
- Experimental procedure for the synthesis of ZnO and ZnO/Ag NPs using Origanum vulgare leaf infusion.
2.2. Synthesis of ZnO and ZnO/Ag NPs
ZnO and ZnO/Ag NPs were synthesized using a combination of green synthesis and the solid-state method. As precursors, zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O, Meyer, 95-100% purity) and silver nitrate (AgNO₃, Meyer, 99% purity) were used, along with a previously prepared infusion of Origanum vulgare leaves. The process began by preheating 100 ml of the infusion to 82°C under constant stirring at 250 rpm (Step 6, Figure 1). Subsequently, the zinc nitrate precursor was added (Step 7), followed by the AgNO₃ precursor in proportions of 0.5, 1, 2, 3, and 8 wt%, depending on the desired composition of the ZnO/Ag system (Step 8). The mixture was stirred continuously for 4 h, during which a pale beige precipitate was formed. Finally, the resulting solution was calcined at 500°C for 4 h (Step 9). Once cooled, it was ground in an agate mortar for further characterization (Step 10). The samples were named M1 for pure ZnO and M2, M3, M4, M5, and M6 for the ZnO/Ag composites, with Ag concentrations of 0.5, 1, 2, 3, and 8 wt%, respectively.
2.3. Antibacterial evaluation
Figure 2 illustrates the experimental procedure used for the antibacterial evaluation of ZnO/Ag samples. The disk diffusion method was employed to evaluate the susceptibility of the G+ bacterium S. aureus (ATCC 25923) and the G- bacterium E. coli (ATCC 25922). Culture media were first sterilized at 15 psi and 120°C for 15 min. Then, 20 mL of medium was poured into each Petri dish and allowed to cool to room temperature (Step 1). Each bacterium was inoculated using a sterile swab moistened with nutrient broth previously incubated for 24 h (Step 2). The inoculum was spread across the agar surface by drawing vertical and horizontal lines, following the disk diffusion method (Step 3). In parallel, disks were prepared using 0.06 g of ZnO/Ag powder, resulting in pellets of 5 mm in diameter and 3 mm in height. The inoculated plates were incubated at 37°C for h (Step 4). Inhibition zones were measured in triplicate for each sample and bacterial strain, and the average values were calculated using ImageJ software. The results for both E. coli and S. aureus are presented in Step 5.
- Schematic of the disk diffusion procedure used to evaluate the antibacterial activity of ZnO/Ag samples against E. coli and S. aureus.
2.4. Characterization
X-ray diffraction (XRD) was utilized to identify the crystalline phases, employing a Bruker D8 Discover diffractometer with Cu-Kα radiation (λ = 1.5418 Å). The equipment operated at 40 kV and 40 mA, covering a 2θ range from 20° to 80°. The crystallite size was calculated using the modified Scherrer equation. Fourier-transform infrared spectroscopy (FTIR) was used to identify the phytochemical constituents present in the samples. A perkin elmer spectrum GX FTIR System was employed, applying the attenuated total reflectance (ATR) technique on dry ZnO and ZnO/Ag powders, within a spectral range of 4000-400 cm⁻1. Morphological analysis was conducted using a JEOL 2010 FEG FASTEM transmission electron microscope (TEM), operated at 200 kV. High-resolution analyses were performed to examine the microstructural effects associated with increasing the percentage of Ag (0.5-8 wt%) in the ZnO matrix. The obtained TEM micrographs were analyzed using ImageJ software version 1.54 to determine the average particle size distribution.
3. Results and Discussion
3.1. Structural analysis by X-Ray diffraction
Figure 3 displays the diffractograms of ZnO samples (M1) and ZnO/Ag with varying Ag concentrations (0.5-8 wt%), corresponding to M2, M3, M4, M5, and M6. The spectra were indexed according to the PDF card 05-0664, representing the hexagonal wurtzite phase (ZnO). Diffraction peaks at 2θ were observed at 31.59°, 34.24°, 36.07°, 47.40°, 56.41°, 62.68°, 66.15°, 67.77°, and 68.89°, associated with the planes (100), (002), (101), (012), (110), (013), (200), (112), and (201), respectively, and are highlighted in black. It was also observed that the diffractograms for samples M4-M6, corresponding to ZnO/Ag, showed additional peaks indexed according to the PDF card 04-0783 for the cubic phase of Ag. Diffraction peaks at 2θ were located at 38.07°, 44.25°, 64.38°, and 77.34°, corresponding to the planes (111), (200), (220), and (311), respectively, marked in red. The crystallite size for each phase was calculated using the modified Scherrer equation, following the method described by Mustapha and Jahan et al. in 2024 [33,34]. The results have been detailed in Table 1.
- Diffraction patterns of the samples (M1-M6).
| Nomenclature | Crystallite size (ZnO) | Crystallite size (Ag) |
|---|---|---|
| M1 | 30.63 | - |
| M2 | 21.78 | 29.66 |
| M3 | 19.60 | 49.08 |
| M4 | 18.36 | 49.41 |
| M5 | 19.24 | 66.06 |
| M6 | 19.91 | 72.26 |
3.2. Structural analysis by Fourier transform infrared spectroscopy
Figure 4 presents the FTIR analysis, which reveals the functional groups present in the ZnO and ZnO/Ag samples. The band at 3430 cm⁻1 corresponds to the stretching vibrations of O-H groups [35]. The absorption band in the range of 2921 cm⁻1 indicates the stretching mode of the C-H bond in methyl groups. Additionally, a band located at 2343 cm⁻1 is observed, attributed to the stretching modes formed due to the O-H group bonding. In the region between 1117 and 1386 cm⁻1 [28], bands corresponding to the stretching vibrations of the C-O bonds are identified. Finally, absorption bands at 400 and 550 cm⁻1 associated with the stretching modes of the Zn-O and Ag-O bonds are identified, confirming the presence of these compounds in the samples [36]. In the case of the Origanum vulgare extract, three main bands are identified. The first band, located at 3430 cm⁻1, corresponds to the hydroxyl (O-H) group with stretching vibrations. Also, the characteristic band of amines, associated with the bending vibrations of the N-H bond (N-H bend), is noted [37]. These bands reflect the phytochemical composition of the extract and its interaction with the synthesized NPs. The assignment of these bands has been summarized in Table 2.
- FTIR spectra of the samples (M1-M6), as well as the spectrum corresponding to the Origanum vulgare extract.
| Band position (cm-1) | Assignment | Description |
|---|---|---|
| 3430 | O-H- | Stretching vibration of hydroxyl groups (O–H) |
| 2921 | C-H | Asymmetric stretching vibration of alkyl C–H bonds |
| 2343 | O-H- | Vibration attributed to bonded O–H groups |
| 1432 | C=C | Stretching vibration of C=C groups |
| 1117-1386 | C-O | Stretching vibrations of C–O bonds |
| 1638 | N-H | Bending vibration of primary amine (N–H) groups |
| 400-500 | Zn-O | Zn–O stretching vibration associated with metal–oxygen bonds |
3.3. Morphological determination by transmission electron microscopy
Figure 5 shows representative EDS spectra of five ZnO/Ag samples, where a progressive increase in the atomic Ag content is observed, ranging from 1.08% in sample (a) to 10.24% in sample (e). This increase is accompanied by changes in the proportions of zinc and oxygen, with a trend toward higher zinc content and reduced oxygen levels. In samples (c) and (d), small amounts of potassium and calcium are also detected, possibly related to residues from the synthesis medium.
- Representative EDS spectra of five ZnO/Ag samples. The spectra correspond to samples M2–M6, shown in ascending order as follows: (a) M2, (b) M3, (c) M4, (d) M5, and (e) M6, where a progressive increase in the atomic Ag content is observed, ranging from 1.08% in sample (a) to 10.24% in sample (e).
Figure 6 displays the morphology and size distribution of samples M1 to M6. In Figures 6(a, b), the results for M1 are shown, where agglomerates composed of quasi-spherical structures with an average size of 50.28 ± 6.5 nm are visible. In contrast, the micrographs for sample M2 show agglomerates of spherical and quasi-spherical structures, with an average size distribution of 22.76 ± 1.58 nm. For sample M3, a greater dispersion among the morphologies (quasi-spherical and faceted particles) is observed, with dimensions ranging from 26.83 ± 1.09 nm, attributed to the increase in the percentage of Ag during the synthesis process. For sample M4 (Figures 6g, h), agglomerates formed by quasi-spherical NPs with an average size distribution of 20.04 ± 1.37 nm are observed. In sample M5, corresponding to an Ag content of 3 wt%, an average particle size distribution of 22.19 ± 0.99 nm was obtained. Finally, sample M6, with an 8 wt% Ag, shows a larger number of agglomerates tending to form spherical and quasi-spherical structures with an average distribution of 19.37 ± 0.28 nm. According to the results presented, the direct influence of Ag concentration on the control of NP size is confirmed.
- Micrographs and graphs showing the average size distribution of ZnO and ZnO/Ag samples at various concentrations: (a, b) M1, (c, d) M2, (e, f) M3, (g, h) M4, (i, j) M5, and (k, l) M6.
3.4. Structural characterization by high-resolution electron microscopy
Figure 7 presents the crystallinity analysis obtained using HRTEM for all samples (M1-M6). The crystallographic planes and their interplanar distances corresponding to ZnO were identified in accordance with the PDF card no. 05-0664, used as a reference. Furthermore, a detailed HRTEM analysis was conducted to assess the effect of increasing the Ag wt% precursor in the ZnO/Ag nanocomposite. In Figures 7(a, b), corresponding to sample M1, crystallographic planes (hkl) were identified using fast fourier transform (FFT), associated with the hexagonal wurtzite phase (ZnO), with a growth direction of [010]. Figures 7(c, d), corresponding to sample M2, display spherical structures also with a growth [010] direction, and a set of crystallographic planes belonging to the wurtzite phase, identified through FFT. In Figures 7(e, f), corresponding to sample M3, NPs with a growth direction along the [010] zone axis are observed. FFT analysis allowed for identifying the interplanar distances and crystallographic planes characteristic of the hexagonal phase of ZnO. Similarly, Figures 7(g, h), corresponding to sample M4, show crystallographic planes associated with the hexagonal phase of ZnO, identified through HRTEM and FFT. In Figures 7(i, j), corresponding to sample M5, the NPs exhibit spherical and semispherical shapes, with a growth direction of [010]. FFT analysis enabled the identification of the characteristic crystallographic planes of ZnO. Finally, Figures 7(k, l), corresponding to sample M6, displays larger structures along with smaller particles. HRTEM and FFT analysis confirmed the presence of crystallographic planes corresponding to the wurtzite phase in all samples, from M1 to M6.
- HRTEM and FFT analysis of samples (a, b) M1, (c, d) M2, (e, f) M3, (g, h) M4, (I, j) M5 and (k, l) M6.
3.5. Evaluation of antibacterial performance as a function of Ag concentration
Table 3 presents the results of antibacterial assays performed on ZnO (M1) and ZnO/Ag (M2-M6) samples, evaluating their ability to inhibit the growth of two bacterial strains: S. aureus and E. coli. The inhibition zones (measured in mm) were recorded in triplicate (halo 1, halo 2, and halo 3), and the mean value with the standard deviation is reported for each condition.
| Sample | Bacteria | Halo 1 (mm) | Halo 2 (mm) | Halo 3 (mm) | Average halo (mm) | Standard deviation |
|---|---|---|---|---|---|---|
| M1 | S. aureus | 12.96 | 12.03 | 12.16 | 12.38 | 0.50 |
| E. coli | 48.46 | 44.58 | 43.93 | 45.66 | 2.45 | |
| M2 | S. aureus | 12.6 | 12.23 | 11.78 | 12.2 | 0.41 |
| E. coli | 27.21 | 26.28 | 31.5 | 28.33 | 2.78 | |
| M3 | S. aureus | 13.05 | 12.35 | 11.88 | 12.42 | 0.59 |
| E. coli | 27.35 | 27.17 | 33.46 | 29.33 | 3.58 | |
| M4 | S. aureus | 13.38 | 12.8 | 13 | 13.06 | 0.29 |
| E. coli | 26.77 | 26.87 | 29.04 | 27.56 | 1.28 | |
| M5 | S. aureus | 13.39 | 12.51 | 13.56 | 13.15 | 0.56 |
| E. coli | 28.63 | 28.39 | 30.5 | 29.17 | 1.16 | |
| M6 | S. aureus | 11.99 | 11.19 | 11.96 | 11.71 | 0.45 |
| E. coli | 27.64 | 27.56 | 33.96 | 29.72 | 3.67 |
Against S. aureus, sample M1 (ZnO) showed a moderate inhibition zone of 12.38 mm with a standard deviation of 0.50 mm, corresponding to the intrinsic antibacterial effect of ZnO. In ZnO/Ag samples, a trend of increased inhibition was observed with the addition of Ag up to 2 wt% (sample M4), which exhibited the highest activity (13.06 mm, SD = 0.29 mm). Sample M5 (3 wt% Ag) showed a comparable average inhibition (13.15 mm) but with reduced reproducibility (SD = 0.56 mm). Notably, sample M6 (8 wt% Ag) showed a decline in inhibition (11.71 mm, SD = 0.45 mm), likely due to saturation effects or Ag surface coverage that may limit active sites.
In the case of E. coli, ZnO (M1) exhibited a notably large inhibition zone (45.66 mm, SD = 2.45 mm), indicating high sensitivity of this strain to ZnO. However, upon the incorporation of Ag, a marked reduction in antibacterial activity was observed. Sample M2 showed a significant drop to 28.33 mm (SD = 2.78 mm), and samples M3 through M5 ranged from 27.56 to 29.33 mm on average. Interestingly, sample M6, with the highest Ag content, showed a slight increase in inhibition to 29.72 mm, though with the highest standard deviation (3.67 mm), suggesting inconsistent performance. This contrasting behavior with E. coli may indicate that the addition of Ag does not produce a synergistic effect with ZnO against G- bacteria, or may even interfere with its efficacy at certain concentrations.
Overall, these results demonstrate that while Ag incorporation enhances antibacterial activity against S. aureus up to an optimal concentration (2 wt%), it may not provide the same benefit against E. coli, where pure ZnO already exhibits strong antibacterial performance. The data also highlight the importance of optimizing Ag content to avoid performance reduction due to excessive doping or NP aggregation effects.
The results obtained in this study corroborate the presence of ZnO as well as the green synthesis of Ag NPs using an extract from Origanum vulgare. Furthermore, they allowed for the analysis of their structural, morphological, and functional characteristics related to their antibacterial activity against S. aureus and E. coli, across a wide range of Ag concentrations (0.5, 1, 2, 3, and 8 wt%) within a ZnO matrix. Morphological analysis via TEM and HRTEM revealed a progressive reduction in the size of the NPs as the concentration of Ag increased, with an average size of 50.28 ± 6.5 nm for M1 and 19.37 ± 0.28 nm for M6. This behavior is consistent with findings reported by Kayani et al. [38], where the incorporation of Ag creates surface tension in the crystalline lattice of ZnO, which limits the growth of the crystallites, reducing the dimensions of the NPs.
In this context, a recent study by Mousavi-Kouhi et al. [39] reported the synthesis of Ag-ZnO nanocomposites using Verbascum speciosum extract, where the crystallite size of pure ZnO NPs was approximately 24.7 nm. This value is comparable to those obtained in our study, where the crystallite size for ZnO reached up to 30.63 nm (M1). Additionally, another study conducted by Islam et al. [40] reported the synthesis of Ag-ZnO nanocomposites via the sol-gel method, using different Ag concentrations (3, 5, and 7 wt%). Their XRD analysis revealed a progressive reduction in crystallite size with increasing Ag content, reporting values of 29.84, 25.64, and 22.36 nm, respectively. These results show a trend consistent with our findings, where the crystallite size of ZnO gradually decreased with the addition of Ag, reaching 18.36 nm for sample M4 (2 wt% Ag). Additionally, EDS confirmed the increase of Ag within the samples (Figure 5), showing a progressive rise in atomic percentage as the nominal Ag content increased. This elemental analysis supports the successful incorporation of Ag into the ZnO matrix, correlating with the observed changes in antibacterial activity.
A larger inhibition zone indicates a higher antimicrobial effectiveness of the sample, as it inhibits bacterial growth over a larger area. A smaller or absent inhibition zone suggests lower or no antibacterial activity [41]. In this regard, samples with intermediate concentrations of Ag (M4 and M5) exhibited the largest inhibition zones in S. aureus, reaching average values of 13.06 mm and 13.15 mm, enhancing their efficiency by 5.5% and 6.2% respectively, compared with M1. These results highlight that a moderate concentration of 2 and 3 wt% Ag is sufficient to significantly enhance the activity of the ZnO/Ag system, while higher concentrations, such as in M6 (8 wt%), result in a decrease in activity (11.71 mm), reducing its efficiency by 5.4%. This could be attributed to a potential saturation effect on the surface of the NPs, where an excess of Ag might block the active sites of ZnO, reducing its interaction with bacteria. This behavior is clearly illustrated in Figure 8, which shows the inhibition zones of S. aureus for each sample in relation to their respective particle sizes.
- Inhibition zones of S. aureus and E. coli as a function of ZnO/Ag particle size for samples M1-M6.
In contrast to the results observed against S. aureus, the antibacterial activity of ZnO and ZnO/Ag samples against E. coli followed a different trend. The pure ZnO sample (M1) exhibited the highest inhibition zone (45.66 mm), indicating a strong intrinsic effect of ZnO on this G- strain. Interestingly, the incorporation of silver led to a marked reduction in antibacterial activity, with M2 showing a significant drop to 28.33 mm. Samples M3 to M5 maintained similar levels of inhibition (ranging from 27.56 to 29.33 mm), suggesting that the presence of Ag did not enhance and may have slightly reduced the efficacy against E. coli. Sample M6, with the highest silver content (8 wt%), exhibited a slight increase in average halo size (29.72 mm), although the high standard deviation indicated greater variability and reduced reproducibility. As shown in Figure 8, the trend observed for E. coli contrasts with that of S. aureus, highlighting a strain-dependent response to both the material composition and particle size. These findings suggest that E. coli is more susceptible to ZnO than to ZnO/Ag systems under the tested conditions, possibly due to the interference of Ag with ZnO’s surface reactivity. The data highlight a strain-dependent response to the material and reinforce the importance of tailoring nanomaterial composition according to the target microorganism.
This divergence in response may also stem from the structural and physiological differences between G+ and G- bacteria. S. aureus, lacking an outer membrane, allows greater interaction with Ag⁺ ions and reactive oxygen species (ROS) [42], which are more efficiently generated in samples with moderate Ag content [3]. Conversely, E. coli possesses an outer membrane that acts as an additional barrier, limiting NP penetration and potentially expressing specific resistance mechanisms against Ag [43].
The antimicrobial effect observed can be explained by the action mechanism of ZnO/Ag NPs and their interaction with the bacterial cell membrane (Figure 9). These nanostructures carry a positive charge, which causes their attraction to the negatively charged bacterial membrane. This interaction leads to membrane rupture and intracellular leakage, mainly due to ion release and the generation of ROS, such as singlet oxygen, hydrogen peroxide, hydroxyl radicals, and superoxide radicals. These events ultimately result in cellular apoptosis [28]. Moreover, smaller particles provide a greater reactive surface area, thereby enhancing their antibacterial activity and effectiveness.
- Proposed antibacterial mechanism of ZnO/Ag NPs.
A comparative summary of reported green-synthesized ZnO/Ag nanomaterials and their antibacterial performance is provided in Table 4, allowing a direct comparison with the results obtained in this study.In this context Thatikayala et al. [27] reported an inhibition zone diameter of 19 mm for S. aureus using Tamarindus indica and a green synthesis method, highlighting the importance of optimizing both the concentration of Ag and the synthesis conditions to maximize antibacterial activity. In contrast, the study by Zare et al. [6] used Hymus vulgaris and the hydrothermal method reported an inhibition zone of 15 mm for S. aureus. Although this value is slightly higher than that obtained with our sample M4, the synthesis conditions used in our study (500°C for 4 h and the use of Origanum vulgare) produced smaller particles (∼20 nm for M4 and M5 compared to ∼30 nm reported in their study). This could favor better interaction with bacteria by increasing the reactive surface area.
| Plant or extract | Method | Bacteria used | Thermal treatment | Halo diameter (mm) | Particle size (nm) | Reference |
|---|---|---|---|---|---|---|
| Thymus vulgaris | Hydrothermal | Staphylococcus aureus and Escherichia coli | 180°C for 3 h | 15 mm (S. aureus), lesser effect on E. coli | ZnO: ∼30 nm, Ag: ∼5 nm | [6] |
| Calotropis gigantea | Combustion in solution | Staphylococcus aureus and Escherichia coli | 500°C for 5 min | S. aureus: 14.46 mm, E. coli: 9.58 mm | ZnO: 100-150 nm, Ag: ∼38 nm | [26] |
| Tamarindus indica | Biosynthesis (pulp extraction) | Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa | 600°C for 2 h | B. subtilis: 17 mm, S. aureus: 19 mm, E. coli: 16 mm, P. aeruginosa: 15 mm | ZnO: 50-60 nm, Ag: ∼5 nm | [27] |
Rajith et al. [26] utilized Calotropis gigantea in a solution combustion method, reporting smaller inhibition zones (14.46 mm for S. aureus and 9.58 mm for E. coli). This lower performance can be attributed to the larger particle size obtained (∼100-150 nm), which is significantly larger than ours, reducing the available surface area for bacterial interactions. Additionally, the trend observed in sample M6 (8 wt% Ag), with a reduced halo diameter (11.71 mm) and an efficiency decrease of 5.4%, aligns with previous reports suggesting that excessive concentrations of Ag can alter the antibacterial effect. This underscores the importance of optimizing composition to maximize antibacterial efficacy.
In summary, it is confirmed that the ZnO/Ag NPs synthesized through a combined green synthesis and solid-state approach exhibit antibacterial properties comparable to, or even superior to, those reported in previous studies. Additionally, the use of Origanum vulgare as a reducing and stabilizing agent represents a novel and sustainable alternative for the production of Ag NPs intended for biomedical applications.
4. Conclusions
The synthesis of ZnO/Ag NPs through a combined green synthesis using Origanum vulgare and the solid-state method proved to be efficient, sustainable, and reproducible. Incorporating Ag at moderate concentrations (2-3 wt%) significantly enhanced antibacterial activity against S. aureus, whereas higher concentrations reduced effectiveness, likely due to surface-saturation effects. Structural and morphological analyses confirmed well-defined crystalline phases, reduced particle sizes, and a homogeneous Ag distribution. Notably, antibacterial performance was found to be bacterium-dependent, showing better efficacy against S. aureus than E. coli, likely due to differences in cell wall structure and resistance mechanisms. These findings position the ZnO/Ag system as a promising platform for biomedical applications, although further studies on biocompatibility and cytotoxicity, are still needed.
Acknowledgment
The authors acknowledge the Secretaría de Ciencias, Humanidades, Tecnología e Innovación (SECIHTI) for funding the Frontier Science Project CBF 2023-2024-267, entitled “Synthesis of transition metal-based nanomaterials with magnetic response through physical and chemical processes for potential environmental and biomedical applications”. The authors thank Melanie Villaseñor Cerón for her laboratory support and M.Sc. Jaqueline Cañetas Ortega for her assistance with electron microscopy. The authors also thank SECIHTI for its financial support of the educational PhD program (CVU no. 859174). Finally, D. Sánchez-Campos and I. G. Meza-Pardo express their gratitude to SECIHTI for its academic support through the National Postdoctoral Program.
CRediT authorship contribution statement
A. Munguía-Martín: investigation, methodology, writing – original draft, writing - review & editing and validation. D. Sánchez-Campos: investigation, methodology, writing – original draft, writing - review & editing and validation. J. Arenas-Alatorre: writing - review & editing, validation, methodology and data curation. I. G. Meza-Pardo: writing - review & editing and validation. D. Mendoza-Anaya: writing - review & editing, validation and data curation. A. Jiménez-González: writing - review & editing, methodology, validation, data curation and V. Rodríguez-Lugo: supervision, project administration, resources, writing – original draft, writing - review & editing.
Declaration of competing interest
The authors have no relevant financial or non-financial interests to disclose.
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Declaration of generative AI and AI-assisted technologies in the writing process
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
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