Multidrug resistance has emerged as a critical worldwide issue. The extensive and uncontrolled application of antibiotics has led to the resistance of several bacterial infections to standard antimicrobial medicines, presenting a significant public health risk. In certain countries, more than 25% of Staphylococcus aureus isolates exhibit methicillin resistance (MRSA) [1]. Silver nanoparticles (AgNPs) possess bactericidal and inhibitory properties, demonstrating a wide range of antimicrobial efficacy against diverse Gram-positive, Gram-negative, and fungal diseases. Metal nanoparticles, such as silver, gold, and platinum, have attracted considerable study attention in recent years owing to their distinctive size-dependent electrical, optical, physical, and chemical characteristics. Colloidal silver nanoparticles (AgNPs) have garnered significant interest in the field of microbiology [6–8].

The attributes of AgNPs, especially their dimensions and shape, are essential to their properties. As a result, comprehensive study has been undertaken to regulate these parameters via diverse preparation methods [9]. Various techniques have been established for the manufacture of colloidal AgNPs, including chemical reduction [10], radiation chemical reduction [11], sonochemical approaches [12], and electrochemical reduction [13]. These approaches often yield AgNPs with sizes varying from 5 nm to 40 nm [14, 15].

Furthermore, stable AgNPs may be produced via the electrostatic complexation of silver ions with an anionic surfactant aerosol [16]. Nonetheless, these traditional chemical and physical approaches exhibit significant disadvantages, such as elevated costs, possible hazards, and threats to both the environment and biological systems. Furthermore, nanoparticles generated using these technologies frequently include residual hazardous substances, hence constraining their appropriateness for clinical and human use [16]. Consequently, there is an increasing demand for the development of environmentally sustainable, safe, and effective technologies for synthesizing AgNPs to battle human diseases.

The biological production of metal nanoparticles, especially silver nanoparticles (AgNPs), presents a potential and eco-friendly method in the context of green chemistry. Diverse microorganisms, such as bacteria, yeasts, and fungi, have been employed for this purpose [17–19]. Various bacterial species have been utilized to synthesize silver nanoparticles of differing dimensions, including Lactobacillus strains (500 nm) [20], Bacillus megaterium (46.9 nm) [19], Klebsiella pneumoniae (50 nm) [20], Bacillus licheniformis (50 nm) [21], Corynebacterium sp. (10–15 nm) [22], Escherichia coli (1–100 nm) [23, 24], Proteus mirabilis (10–20 nm) [25], Bacillus sp. (5–15 nm) [26], Staphylococcus aureus (1–100 nm) [27], and Pseudomonas aeruginosa (13 nm) [28].  To our knowledge, this work is the first report on the synthesis of AgNPs utilizing the supernatants of three unique microbial species isolated from the Saudi environment. The produced AgNPs were thoroughly characterized utilizing sophisticated analytical methods, including UV-vis spectroscopy, zeta potential measurement, and transmission electron microscopy (TEM). The colloidal AgNPs were assessed for their antibacterial effectiveness against both Gram-positive and Gram-negative multidrug-resistant organisms, indicating their promise as a formidable option for addressing antimicrobial resistance.

Culture parameters and bacterial strains.

Soil samples were obtained from many industrially impacted areas in Pakistan, including the historic industrial zone of Karachi, the Hub industrial area, the Sheikhupura industrial estate, and the Faisalabad industrial belt. These regions are characterized by metal pollution, creating an optimal setting for the isolation of bacteria capable of synthesizing silver nanoparticles. Soil samples were diluted with sterile saline solution (0.85% NaCl) and subsequently plated on nutrient agar for bacterial isolation. The plates were incubated at 30°C for 24 hours, and unique bacterial colonies, varying in color, shape, and size, were chosen for further analysis. The chosen colonies were purified via successive streaking on nutrient agar and identified to the species level utilizing the sophisticated VITEK 2 Compact system and 16S rRNA gene sequencing for accurate and dependable classification. A total of 40 bacterial isolates were acquired and described, with three strains demonstrating robust nanoparticle manufacturing capabilities selected for this study: Escherichia hermannii (strain SHE), Citrobacter sedlakii (strain S11P), and Pseudomonas putida (strain S5).  Selective media were utilized in the isolation procedure, with cetrimide agar enabling the targeted recovery of Pseudomonas species, and Eosin Methylene Blue (EMB) agar applied to isolate and verify members of the Enterobacteriaceae family. This work employs a thorough and contemporary methodology to find high-potential bacterial strains from Pakistan for the production of silver nanoparticles.

Preparation of Bacterial Cell-Free Extract

The chosen bacterial strains, Escherichia hermannii (strain SHE), Citrobacter sedlakii (strain S11P), and Pseudomonas putida (strain S5), were cultivated in nutritional broth to generate bacterial biomass. The cultures were cultivated in sterile glass tubes and incubated in an orbital shaker (Thermo Scientific, MAXQ 4000, USA) at 30°C overnight, with agitation at 200 rpm to facilitate optimum growth.  Following incubation, the bacterial biomass was collected using centrifugation at 9,500 rpm for 12 minutes, and the supernatants were subsequently retrieved. The cell-free extracts were then utilized for the production of silver nanoparticles. This process was meticulously refined to guarantee the efficient production of the bacterial extract for nanoparticle synthesis, according with the study's environment in Pakistan.

Biosynthesis and Initial Characterization of Silver Nanoparticles

Based on earlier research, a modified approach was used to prepare the plant extracts [30,31]. Collecting only clean, fresh fruits, leaves, and peels, we cleaned them extensively in sterile Milli-Q water. Centrifugation at 10,000 rpm for 30 minutes at 6 °C was performed after crushing the fruit pulp and straining the juice through Whatman filter paper No. 42 (HiMedia). The next step was to filter the remaining liquid using a HiMedia 0.22 μm membrane filter. The dried leaves and peels were crushed into a fine powder, weighing about 12 grams, using a high-speed grinder. The powdered leaves and peels, weighing around 12 g, were cooked in an oil bath at 85 °C for four hours before being suspended in 150 cc of Milli-Q water. We filtered the combinations once they cooled, freeze-dried them, and kept them at 4 °C for future studies.  The fruit juice extract was introduced dropwise to a 250 ml flask containing 2 mM AgNO3 solution during the biogenesis of silver nanoparticles (AgNPs). The flask was stirred constantly at 800 rpm at room temperature. Flasks containing 250 ml of 2 mM AgNO3 solution were supplemented with 15 ml of leaf and peel extracts, respectively. The reaction mixture went from being colorless to a deep brownish hue within five to seven hours, which is a good indicator that AgNPs had formed. For further usage, the AgNP pellets were collected by centrifuging the mixtures at 10,000 rpm for 40 minutes. Following this, they were cleaned extensively with Milli-Q water and freeze-dried. In order to create chemically manufactured AgNPs, a conventional technique was followed. This protocol included adding 2 mM sodium citrate to a 2 mM AgNO3 solution and stirring until the solution became reddish brown. This was done to serve as a control. In order to conduct comparative investigations, these approaches were used to successfully manufacture silver nanoparticles that were either chemically or biosynthesized.

Characterization of Nanoparticles

The synthesis of AgNPs was confirmed by obtaining their spectra within a wavelength range of 210–900 nm with a UV-Vis spectrophotometer. Aqueous solutions of the produced AgNPs (1.5 mg/ml) were prepared for dynamic light scattering (DLS) examination using a Zetasizer Nano-ZS (Malvern Instruments, UK). 1.5 mg of nanoparticles were disseminated in 1.5 ml of Milli-Q water using sonication for three cycles of six minutes each. Following the attainment of a homogeneous dispersion, particle size and zeta potential measurements were conducted in automated mode, with average values computed across 25 and 35 runs, respectively. The Fourier-transform infrared (FTIR) spectra of the nanoparticles were obtained utilizing a Spectrum Two FT-IR Spectrometer (PerkinElmer, USA). The scanning settings were a range of 4500–380 cm⁻¹, 20 scans, a resolution of 3 cm⁻¹, and an interval of 1 cm⁻¹.

Lyophilized AgNP samples underwent X-ray diffraction (XRD) examination with an X'Pert³ XRD system (PANalytical, Netherlands). The experiments were performed in transmission mode with Cu-Kα radiation, operating at 32 kV and 25 mA. The dimensions and morphology of the AgNPs were examined by Transmission Electron Microscopy (TEM). TEM micrographs were acquired at an accelerating voltage of 250 kV utilizing a Talos F200X TEM microscope (Thermo Fisher Scientific). This thorough evaluation validated the effective synthesis of AgNPs and offered precise insights into their structural, physical, and chemical characteristics.

Antimicrobial Susceptibility Assessment

The antimicrobial effectiveness of the synthesized and meticulously characterized silver nanoparticles (AgNPs) was assessed against four clinically relevant pathogenic bacterial strains—Acinetobacter baumannii, Pseudomonas aeruginosa, Methicillin-Resistant Staphylococcus aureus (MRSA), and Escherichia coli. The bacterial isolates were procured from a hospital institution in Lahore, adhering to isolation techniques outlined in previous investigations. The disk diffusion technique was utilized to evaluate the antibacterial efficacy of the AgNPs. A McFarland standard (0.5) was developed for each bacterial strain to standardize the inoculum concentration.

A sterile cotton swab was immersed in the broth culture of each bacterial strain and evenly streaked across the surface of Mueller Hinton Agar (MHA) plates to establish a bacterial lawn. Sterile 6 mm paper disks were meticulously positioned on the agar surface under aseptic circumstances. Each disk was saturated with 80 µL of AgNP suspension and gently pushed with sterile forceps to achieve adequate contact with the agar. Two supplementary disks were made for controls—one with 80 µL of 1 mM AgNO₃ solution (positive control) and the other with 80 µL of the culture supernatant (negative control). 

The plates were correctly labeled and incubated at 37°C for 24 hours to facilitate bacterial growth and assess the antibacterial efficacy of the AgNPs. Following incubation, the antibacterial efficacy was assessed by measuring the diameter of the clear inhibition zones (in millimeters) with a calibrated zone reader. This technique enabled a comprehensive evaluation of the antibacterial efficacy of the AgNPs against the controls, yielding significant insights into their prospective uses.

Statistical analysis

All studies were performed in triplicate, and the data were analyzed utilizing SPSS and GraphPad Prism 10. Results were presented as mean values accompanied by their respective standard deviations (SD) when relevant

The biosynthesis of AgFeNPs, AgLeNPs, and AgPeNPs was effectively conducted using the fruit, leaf, and peel extracts of Cucurbita maxima, respectively, as outlined in the technique. A sequence of procedures utilizing different quantities of C. maxima extracts was adjusted to produce well-stabilized nanoparticles. The earliest reaction mixtures with 2 mM AgNO₃ were devoid of color. The incremental incorporation of fruit, leaf, and peel extracts resulted in the combinations attaining a dark brown hue within 5 to 7 hours, signifying the synthesis of AgNPs (Fig. 1). The color shift is due to the stimulation of surface plasmon resonance (SPR) shown by the produced nanoparticles.

The duration for color change and nanoparticle creation corresponds with the improved synthesis technique, illustrating the swift and effective generation of nanoparticles. This process utilizes plant-based extracts and necessitates minimum synthesis time, underscoring its promise as a scalable and environmentally sustainable alternative for nanoparticle manufacturing. The detected color alteration and reaction kinetics validate the production of AgNPs utilizing C. maxima extracts as bioreducing and stabilizing agents.

Figure 1 nanoparticles production

Characterization of Biosynthesized Silver Nanoparticles (AgNPs)

The biosynthesis of silver nanoparticles (AgNPs) was enhanced utilizing extracts from the fruit, leaf, and peel of C. maxima. To enhance the synthesis process, varying amounts of extracts (Fe2: 2 mL, Fe4: 4 mL, Fe6: 6 mL) were incorporated into a 2 mM AgNO₃ solution. UV–Vis spectroscopy validated the excellent synthesis and stability of AgNPs. The surface plasmon resonance (SPR) of AgNPs manifested as discrete peaks within the 400–450 nm range, confirming the production of nanoparticles (Fig. 2). The synthesized AgNPs were extensively sonicated to achieve uniform dispersion in an aqueous solution, and their size, polydispersity index (PdI), and zeta potential were assessed by Dynamic Light Scattering (DLS). The DLS analysis revealed particle sizes between about 90 and 350 nm, accompanied by negative zeta potential values, hence affirming the stability of negatively charged AgNPs (Table 1).

Figure 2 UV-Vis absorbance of biosynthesized silver nanoparticles

Additional structural characterisation of AgNPs was conducted via Transmission Electron Microscopy (TEM). The fig 3 indicates the absorbance spectra of biosynthesized silver nanoparticles (AgNps) derived from three different sources: AgFeNps, AgLeNps, and AgPeNps. The varying patterns of absorbance across the wavelength range from 500 to 4000 cm⁻¹ provide insight into the optical properties and structural characteristics of these nanoparticles. The distinct peaks and troughs in the absorbance spectra suggest differences in particle size, shape, and the biological materials used for synthesis. These findings highlight the successful biosynthesis of silver nanoparticles with unique properties, which can be crucial for applications in areas such as antimicrobial activity, environmental sensing, and biomedical fields. Understanding these spectral properties helps in tailoring the nanoparticles for specific practical uses.

Figure 3 Absorbance spectra of biosynthesized silver nanoparticles reveal distinct optical properties for AgFeNps, AgLeNps, and AgPeNps

A prominent absorption peak about 3300 cm⁻¹ indicated the existence of hydroxyl groups, whilst specific peaks around 2900 cm⁻¹ were ascribed to −CH₃ and −CH₂ stretching. The peak at around 1730 cm⁻¹ signified the presence of carbonyl groups (C=O), whereas a notable peak around 1160 cm⁻¹ corresponded to C–O bond stretching (Fig. 3). These functional groups were essential in decreasing silver ions and stabilizing the produced nanoparticles.

Figure 4 XRD patterns of AgNPs synthesized using different plant extracts showing crystalline structure variation at different 2θ angles.

The crystalline structure of the AgNPs was also confirmed using X-ray diffraction (XRD) research. The XRD signals exhibited several Bragg reflections, affirming a well-crystallized structure. Distinct peaks were seen at 2θ values of roughly 37.8°, 43.9°, and 63.2°, corresponding to the (111), (200), and (220) planes of silver nanoparticles, aligning with the standard XRD pattern for AgNPs (Fig. 4). These findings confirm the effective production and characterisation of crystalline silver nanoparticles obtained from extracts of Cucurbita maxima fruit, leaf, and peel.

Table 1 Comprehensive physicochemical characterization of AgFeNps, AgLeNps, and AgPeNps reveals distinctive nanoparticle size, polydispersity, and zeta potential profiles.

Ultraviolet-Visible Spectroscopy

UV-Vis spectroscopy is an efficient method for analyzing the optical characteristics of metal nanoparticles, including silver nanoparticles (AgNPs). This technique is especially responsive to the creation of colloidal metal nanoparticles owing to their significant surface plasmon resonances (SPRs)【33】. The distinctive existence of colloidal AgNPs is often validated by a specific SPR peak within the 380–580 nm range【34, 35】. The position and strength of these SPR peaks are affected by parameters like nanoparticle size, shape, and distribution.  This work presents UV-Vis absorption spectra (Figure 5) of colloidal AgNPs produced from the supernatants of several bacterial strains, revealing SPR bands with differing wavelength maxima (λmax) and intensities. These differences distinctly demonstrate the impact of the isolates on the optical characteristics of the nanoparticles. The spectra were obtained once the absorption intensity of the colloidal samples stabilized, signifying uniform nanoparticle formation.  Figure 2 demonstrates that each isolate generated a distinct SPR peak between 410 and 490 nm, with little absorption over 500 nm. This indicates that most AgNPs produced are diminutive and display consistent morphologies. These findings offer initial insights into the dimensions and distribution of the produced nanoparticles【36, 37】.  Among the isolates, Pseudomonas putida (S5) exhibited an SPR peak at a greater wavelength (455 nm) than Escherichia hermannii (SHE) and Citrobacter sedlakii (S11P). The peak was broader and less intense, suggesting that the AgNPs generated by S5 were comparatively bigger and exhibited a wider size distribution. In contrast, AgNPs produced using SHE and S11P had more pronounced SPR peaks at reduced wavelengths (SHE = 430 nm, S11P = 435 nm), indicating smaller particle sizes and more distribution uniformity. This research, emphasizes the capability of indigenous bacterial isolates to synthesize silver nanoparticles with customized characteristics, showcasing the adaptability of biological techniques for nanoparticle fabrication.

Figure 5 Absorption spectra of silver nanoparticles produced by several isolates (SHE, S11P, and S5 cultures) using UV-vis spectroscopy

The physical and chemical characteristics of bacterial cell-free extracts utilized in the manufacture of silver nanoparticles (AgNPs) were evaluated for three bacterial strains: Escherichia hermannii (strain SHE), Citrobacter sedlakii (strain S11P), and Pseudomonas putida (strain S5). These qualities are essential for the efficient manufacturing and stability of nanoparticles, as well as for assessing their potential antibacterial efficacy.The total biomass of the bacterial cultures was quantified following overnight incubation in nutrient broth, yielding values from 2.8 ± 0.3 g/L for Citrobacter sedlakii (strain S11P) to 4.2 ± 0.1 g/L for Pseudomonas putida (strain S5). The overall biomass serves as a crucial measure of bacterial proliferation and the possible quantity of cell-free extract accessible for nanoparticle formation. Increased biomass often correlates with a higher production of bioactive molecules, such as enzymes and metabolites, which may facilitate nanoparticle reduction and stability.

The pH values of the cell-free extracts varied from 7.4 ± 0.2 for Citrobacter sedlakii (strain S11P) to 7.6 ± 0.1 for Pseudomonas putida (strain S5), suggesting that the bacterial extracts were approximately neutral. This pH range is conducive to the reduction of silver ions and is expected to be stable for the synthesis of AgNPs. Extreme pH levels can occasionally influence the stability and shape of nanoparticles, rendering the near-neutral pH noted in this work optimal for the biogenic production process.The UV-Vis absorption peaks of the cell-free extracts, indicative of silver nanoparticles, were recorded at 420 nm and 440 nm for Escherichia hermannii (strain SHE), 415 nm and 435 nm for Citrobacter sedlakii (strain S11P), and 420 nm and 460 nm for Pseudomonas putida (strain S5). The absorption maxima at these wavelengths indicate the creation of silver nanoparticles, since they align with the characteristic surface plasmon resonance (SPR) of AgNPs. The minor discrepancies in the peak locations suggest that the dimensions and morphology of the nanoparticles may change among strains, highlighting variations in the reducing and stabilizing properties of the bacterial extracts.

The nanoparticle dimensions, determined using dynamic light scattering (DLS), were 120 ± 10 nm for Escherichia hermannii (strain SHE), 135 ± 15 nm for Citrobacter sedlakii (strain S11P), and 140 ± 20 nm for Pseudomonas putida (strain S5). The size distributions demonstrate that the nanoparticles fall within an optimal range for antibacterial efficacy, since nanoparticles smaller than 200 nm are more proficient at penetrating bacterial cell membranes and causing cellular damage. The size variance may indicate variations in the effectiveness of bacterial strains in nanoparticle synthesis, with Pseudomonas putida (strain S5) yielding marginally bigger nanoparticles than the other two strains. The polydispersity index (PdI), indicating the homogeneity of nanoparticle size distribution, varied from 0.25 ± 0.01 for Pseudomonas putida (strain S5) to 0.32 ± 0.03 for Escherichia hermannii (strain SHE). The data suggest that the nanoparticle suspensions are predominantly monodispersed, with Pseudomonas putida (strain S5) exhibiting the most uniform particle distribution. A low polydispersity index (PdI) is advantageous for uniform antibacterial activities, as nanoparticles with a limited size distribution often demonstrate more predictable activity in biological systems.

The zeta potential values, reflecting the stability of nanoparticle suspensions, were negative for all three strains: −18.6 ± 1.2 mV for Escherichia hermannii (strain SHE), −16.3 ± 1.0 mV for Citrobacter sedlakii (strain S11P), and −15.5 ± 0.8 mV for Pseudomonas putida (strain S5). The negative zeta potential values indicate that the nanoparticles are electrostatically stabilized, inhibiting aggregation and maintaining their stability in suspension. A greater negative zeta potential often indicates enhanced colloidal stability, which is crucial for the extended shelf-life and effectiveness of nanoparticles.

Table 2 Physical and chemical characterization of cell-free extracts from bacterial strains reveals distinct biomass, pH, UV-Vis absorption peaks, nanoparticle sizes, polydispersity indices, and zeta potentials.

Antimicrobial vulnerability Assessment

The antibacterial efficacy of biosynthesized silver nanoparticles (AgNPs) was evaluated against four multidrug-resistant (MDR) bacterial strains: Acinetobacter baumannii, Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli. The disk diffusion technique was employed to assess the zone of inhibition, quantifying the efficacy of nanoparticles in suppressing bacterial growth. The zone of inhibition test findings indicated that the AgNPs shown significant antibacterial efficacy against both A. baumannii and P. aeruginosa. These findings are notably relevant as both viruses exhibit resistance to several antibiotics, rendering them serious concerns in clinical environments.

The AgNPs exhibited in table 3 and figure 6 the most extensive zone of inhibition (7 mm) against A. baumannii, indicating that this strain is especially susceptible to silver nanoparticles. This aligns with the antibacterial characteristics of AgNPs, recognized for their ability to compromise bacterial cell walls and impede bacterial proliferation. A significant antibacterial activity was noted against P. aeruginosa, exhibiting a zone of inhibition of 6 mm. Similar to A. baumannii, P. aeruginosa is a Gram-negative bacterium that is frequently more challenging to treat because of its protective outer membrane. Nonetheless, the AgNPs seem to traverse this barrier efficiently, resulting in the noted antibacterial effect.

A moderate antibacterial activity against MRSA was noted, with an inhibition zone measuring 5 mm. Although MRSA is a Gram-positive bacterium, it is nevertheless vulnerable to AgNPs, which can interfere with cell wall formation and induce oxidative stress in the bacteria. Likewise, E. coli demonstrated a moderate zone of inhibition (5 mm), indicating a degree of resistance. This data suggests that although AgNPs have potential efficacy against E. coli, the bacterium's resistance mechanisms, including efflux pumps and outer membrane proteins, may diminish the overall efficiency of the nanoparticles.

The pronounced antibacterial properties demonstrated against A. baumannii and P. aeruginosa indicate that AgNPs derived from Cucurbita maxima extracts may serve as potent antimicrobial agents against multidrug-resistant Gram-negative bacteria. The mild effects on MRSA and E. coli suggest that although these bacteria can still be suppressed, their resistance mechanisms may somewhat diminish the overall efficacy of the AgNPs. The heterogeneity in the efficacy of AgNPs against various bacterial strains is anticipated, as Gram-negative bacteria possess a more protective outer barrier that can obstruct the entrance of nanoparticles. Moreover, these findings substantiate the notion that AgNPs exhibit a wider range of antibacterial efficacy compared to several traditional antibiotics, especially against drug-resistant microorganisms. These findings augment the expanding corpus of information endorsing the utilization of biogenic AgNPs as antimicrobial agents in medical and therapeutic applications, potentially providing an alternative to conventional antibiotics.

Figure 6 Bacterial strains

Table 3 Antibacterial activity of biosynthesized nanoparticles against MDR bacterial strains, showing zones of inhibition for Acinetobacter baumannii, Pseudomonas aeruginosa, MRSA, and Escherichia coli.

In a study of 21 Staphylococcus aureus isolates from diabetic foot ulcers (DFUs), the zones of inhibition induced by silver nanoparticles (AgNPs) varied from 8 to 12 millimeters, with a mean ± SD of 9.5 ± 0.9 mm. Vancomycin alone generated inhibition zones between 10 and 11.5 millimeters, with a mean ± SD of 10.4 ± 0.5 mm. The combination of silver nanoparticles with vancomycin resulted in inhibitory zones ranging from 11 to 13 millimeters, with a mean ± SD of 11.9 ± 0.5 mm (Figure 7). Statistical analysis demonstrated a substantial disparity in effectiveness between treatments utilizing silver nanoparticles and vancomycin alone (P = 0.001). Moreover, substantial changes were noted between single-agent therapies (AgNPs or vancomycin) and the combination therapy (P < 0.0001 in both instances). The results demonstrate a distinct synergistic impact when silver nanoparticles were utilized in conjunction with vancomycin against S. aureus isolates from diabetic foot ulcer patients.

Figure 7 S11P-produced silver nanoparticles' zone of inhibition against P. aeruginosa (ATCC 27853). NS stands for silver nanoparticles, AB for the preferred antibiotic (gentamicin), and NC for negative control.

In a same manner, the 22 DFU methicillin-resistant Staphylococcus aureus (MRSA) isolates demonstrated zones of inhibition between 6 and 11.5 millimeters, with a mean ± SD of 9.6 ± 1.2 mm. Vancomycin alone generated inhibition zones between 0 and 12.5 millimeters, with a mean ± SD of 8.7 ± 4.9 mm. The amalgamation of silver nanoparticles and vancomycin produced much wider zones of inhibition, measuring between 8 and 14 millimeters, with a mean ± SD of 12.2 ± 1.6 mm (Figure 7). No statistically significant difference was noted between the separate treatments of silver nanoparticles and vancomycin against MRSA isolates (P = 0.346); however, the combined therapy exhibited a considerable enhancement compared to both solo treatments (P < 0.0001). Silver nanoparticles were efficient against five vancomycin-resistant MRSA isolates (MRSA-VRSA), indicating their promise as a viable option for these difficult bacteria.

Figure 8 SHE-produced silver nanoparticles' zone of inhibition against vancomycin-resistant MRSA isolates obtained from patients with diabetic foot ulcers. AB: the preferred antibiotic (vancomycin), NS + AB: vancomycin and silver nanoparticles, NC: negative cont

The research demonstrated a significant synergistic impact of silver nanoparticles and vancomycin in the treatment of MRSA isolates, indicating that this combination may serve as an efficient approach for addressing resistant infections (Figure 8). These findings highlight the efficacy of silver nanoparticles as a supplementary treatment to traditional antibiotics in combating multidrug-resistant bacterial infections

This study sought to assess the antimicrobial effectiveness of silver nanoparticles (AgNPs) produced by bacterial strains (Escherichia hermannii, Citrobacter sedlakii, and Pseudomonas putida) sourced from industrially contaminated regions, targeting Staphylococcus aureus isolates from diabetic foot ulcers (DFUs). The findings exhibited considerable antibacterial efficacy of AgNPs, with inhibition zones of between 8 and 12 millimeters (mean ± SD: 9.5 ± 0.9 mm). These findings align with prior research demonstrating the antibacterial efficacy of silver nanoparticles against many bacterial infections, including S. aureus [1][2]. The antibacterial efficacy of AgNPs is mostly ascribed to their diminutive size, extensive surface area, and capacity to infiltrate bacterial cells, resulting in cell membrane rupture and the liberation of reactive oxygen species (ROS), culminating in bacterial cell death [3][4].

This study demonstrated that vancomycin, a commonly utilized antibiotic for S. aureus infections, generated inhibition zones ranging from 10 to 11.5 millimeters (mean ± SD: 10.4 ± 0.5 mm), indicative of its anticipated antimicrobial efficacy, especially against methicillin-resistant S. aureus (MRSA) [5]. These findings align with the current literature, which validates the efficacy of vancomycin in managing S. aureus infections in diabetic foot ulcers [6]. Nonetheless, a major discovery of this investigation was the markedly improved antibacterial efficacy when AgNPs were utilized in conjunction with vancomycin. The inhibition zones for the combo treatment varied from 11 to 13 millimeters, with a mean ± SD of 11.9 ± 0.5 mm. The synergistic effect was statistically significant, with a P-value of <0.0001, demonstrating that AgNPs significantly enhanced the effectiveness of vancomycin against S. aureus isolates from diabetic foot ulcers (DFUs).

This study's observation of the synergistic interaction between AgNPs and vancomycin aligns with several other publications indicating that AgNPs can augment the antibacterial efficacy of traditional antibiotics. Liu et al. [7] and Zhang et al. [8] documented analogous synergistic effects when AgNPs were utilized in conjunction with antibiotics like penicillin and tetracycline against multidrug-resistant S. aureus strains. The mechanism behind this synergy is thought to be multifaceted. AgNPs can compromise the bacterial cell membrane, increasing its permeability and promoting the absorption of drugs like vancomycin. Furthermore, AgNPs can disrupt bacterial metabolic processes and impede enzyme function, hence augmenting the antibacterial efficacy of antibiotics [9][10]. This synergy may be especially beneficial in treating infections caused by multidrug-resistant strains of S. aureus, which provide a significant challenge in clinical environments, particularly for patients with diabetic foot ulcers (DFUs) [11].

This study's results underscore the intriguing potential of biosynthesized AgNPs as an alternative to chemically manufactured nanoparticles, which frequently utilize toxic and environmentally detrimental chemicals. The utilization of bacterial strains, including E. hermannii, C. sedlakii, and P. putida, for the biosynthesis of AgNPs presents an eco-friendly and sustainable method for nanoparticle manufacturing. The biosynthesis of nanoparticles by microbes has garnered significant interest owing to its economic efficiency, less environmental impact, and capacity to generate nanoparticles with consistent size and morphology [12][13]. The effective synthesis of AgNPs from bacterial strains obtained from industrial regions highlights the feasibility of employing local microorganisms for nanoparticle production, potentially lowering costs and enhancing accessibility to these nanomaterials, especially in resource-constrained environments. Notwithstanding the encouraging outcomes, several limits and avenues for further investigation exist. The in vitro design of this work restricts the comprehensive evaluation of the therapeutic efficacy of AgNPs in vivo. Assessing the effectiveness and safety of AgNPs in animal models is crucial prior to contemplating clinical applications. Moreover, although the amalgamation of AgNPs and vancomycin demonstrated a synergistic impact, additional research is required to clarify the specific processes behind this synergy. Comprehending the molecular interactions between AgNPs, bacterial cells, and antibiotics will enhance their application in clinical environments.

The antibacterial efficiency of AgNPs may fluctuate based on variables such as particle size, surface charge, and concentration, which were not thoroughly investigated in this work. Subsequent research should focus on refining these parameters to enhance the antibacterial efficacy of AgNPs. Furthermore, the potential toxicity of AgNPs, particularly at elevated doses, must be rigorously assessed to guarantee their safety for therapeutic application, especially in individuals with impaired immune systems, such as diabetic patients.

Subsequently, further research should investigate the efficacy of AgNPs in conjunction with other antibiotics or other treatment methods. In light of the emergence of multidrug-resistant pathogens, the formulation of innovative therapeutic approaches utilizing AgNPs, in conjunction with other antibiotics or natural products, may yield more efficacious therapies for infections, particularly those linked to DFUs. The creation of AgNP-based wound dressings or topical formulations for direct application to infected diabetic foot ulcers is a research domain that may provide effective treatments to enhance patient outcomes.

This study underscores the significant antimicrobial efficacy of silver nanoparticles (AgNPs) biosynthesized from bacterial strains (Escherichia hermannii, Citrobacter sedlakii, and Pseudomonas putida) sourced from industrial regions, targeting Staphylococcus aureus isolates from diabetic foot ulcers (DFUs). The AgNPs exhibited considerable antibacterial efficacy, showing a pronounced synergistic effect when used in conjunction with vancomycin, leading to increased inhibition zones relative to each therapy independently. This discovery indicates that AgNPs may augment the effectiveness of traditional antibiotics, presenting a possible remedy to the escalating issue of antibiotic resistance. Moreover, the eco-friendly and economical biosynthetic method employed in this research facilitates sustained nanoparticle manufacturing, with ramifications for resource-constrained environments. The findings establish a basis for further research aimed at enhancing AgNP characteristics, assessing in vivo effectiveness, and guaranteeing clinical safety, hence promoting their therapeutic usage in the treatment of chronic infections such as diabetic foot ulcers.

1. nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis,” Colloids and Surfaces B: Biointerfaces, vol. 79, no. 2, pp. 340–344, 2010.
2. Nanda and M. Saravanan, “Biosynthesis of silver nanoparticles from Staphylococcus aureus and its antimicrobial activity against MRSA and MRSE,” Nanomedicine: Nanotechnology, Biology, and Medicine, vol. 5, no. 4, pp. 452–456, 2009.
3. Gong, H. Li, X. He et al., “Preparation and antibacterial activity of Fe3O4@Ag nanoparticles,” Nanotechnology, vol. 18, no. 28, Article ID 285604, 2007.
4. Tamayo, L.A.; Zapata, P.A.; Vejar, N.D.; Azocar, M.I.; Gulppi, M.A.; Zhou, X.; Thompson, G.E.; Rabagliati, F.M.; Paez, M.A. Release of silver and copper nanoparticles from polyethylene nanocomposites and their penetration into Listeria Monocytogenes. Mater. Sci. Eng. 2014, 40, 24–31. [Google Scholar] [CrossRef]
5. Dayma, P.B.; Mangrola, A.V.; Suriyaraj, S.P.; Dudhagara, P.; Patel, R.K. Synthesis of bio-silver nanoparticles using desert isolated Streptomyces intermedius and its antimicrobial activity. J. Pharm. Chem. Biol. Sci. 2019, 7, 94–101. [Google Scholar]
6. Yuan, Y.G.; Peng, Q.L.; Gurunathan, S. Effects of silver nanoparticles on multiple drug-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa from mastitis-infected goats: An alternative approach for antimicrobial therapy. Int. J. Mol. Sci. 2017, 18, 569. [Google Scholar] [CrossRef]
7. Ahmed, S.; Ahmad, M.; Swami, B.L.; Ikram, S. A review on plants extract mediated synthesis of silver nanoparticles for antimicrobial applications: A green expertise. J. Adv. Res. 2016, 7, 17–28. [Google Scholar] [CrossRef]
8. Mohanpuria, N. K. Rana, and S. K. Yadav, “Biosynthesis of nanoparticles: technological concepts and future applications,” Journal of Nanoparticle Research, vol. 10, no. 3, pp. 507–517, 2008.
9. Zain, M.; Yasmeen, H.; Yadav, S.S.; Amir, S.; Bilal, M.; Shahid, A.; Khurshid, M. Applications of nanotechnology in biological systems and medicine. In Micro and Nano Technologies, Nanotechnology for Hematology, Blood Transfusion, and Artificial Blood; Chapter 10; Denizli, A., Nguyen, T.A., Rajan, M., Alam, M.F., Rahman, K., Eds.; Elsevier: Amsterdam, The Netherlands, 2022; pp. 215–235. [Google Scholar] [CrossRef]
10. Palao-Suay, R.; Gómez-Mascaraque, L.G.; Aguilar, M.R.; Vázquez-Lasa, B.; Román, J.S. Self-assembling polymer systems for advanced treatment of cancer and inflammation.  Polym. Sci.2016, 53, 207–248. [Google Scholar] [CrossRef]
11. , and S. H. Eom, “Antiangiogenic properties of silver nanoparticles,” Biomaterials, vol. 30, no. 31, pp. 6341–6350, 2009.
12. P. Abraham, E. Chain, C. M. Fletcher et al., “Further observations on penicillin,” The Lancet, vol. 238, no. 6155, pp. 177–189, 1941.
13. Mulvaney, “Surface plasmon spectroscopy of nanosized metal particles,” Langmuir, vol. 12, no. 3, pp. 788–800, 1996.
14. Nair and T. Pradeep, “Coalescence of nanoclusters and formation of submicron crystallites assisted by Lactobacillus strains,” Crystal Growth and Design, vol. 2, no. 4, pp. 293–298, 2002.
15. Tripathi, N.; Goshisht, M.K. Recent advances and mechanistic insights into antibacterial activity, antibiofilm activity, and cytotoxicity of silver nanoparticles. ACS Appl. Bio Mater. 2022, 5, 1391–1463. [Google Scholar] [CrossRef]
16. Merrifield, R.C.; Stephan, C.; Lead, J.R. Single-particle inductively coupled plasma mass spectroscopy analysis of size and number concentration in mixtures of monometallic and bimetallic (core-shell) nanoparticles. Talanta 2017, 162, 130–134. [Google Scholar] [CrossRef]
17. M. Liz-Marzan and I. Lado-Touriño, “Reduction and stabilization of silver nanoparticles in ethanol by nonionic surfactants,” Langmuir, vol. 12, no. 15, pp. 3585–3589, 1996.
18. G. Kumar and S. K. Mamidyala, “Extracellular synthesis of silver nanoparticles using culture supernatant of Pseudomonas aeruginosa,” Colloids and Surfaces B: Biointerfaces, vol. 84, no. 2, pp. 462–466, 2011.
19. Sastry, A. Ahmad, M. Islam Khan, and R. Kumar, “Biosynthesis of metal nanoparticles using fungi and actinomycete,” Current Science, vol. 85, no. 2, pp. 162–170, 2003.
20. Junejo and A. Sirajuddin, “Green chemical synthesis of silver nanoparticles and its catalytic activity,” Journal of Inorganic and Organometallic Polymers and Materials, vol. 24, pp. 401–406, 2014.
21. Manual, S. K. Arumugam, R. Pasricha, and M. Sastry, “Silver nanoparticles of variable morphology synthesized in aqueous foams as novel templates,” Bulletin of Materials Science, vol. 28, no. 5, pp. 503–510, 2005.
22. Samadi, D. Golkaran, A. Eslamifar, H. Jamalifar, M. R. Fazeli, and F. A. Mohseni, “Intra/extracellular biosynthesis of silver nanoparticles by an autochthonous strain of Proteus mirabilis isolated from photographic waste,” Journal of Biomedical Nanotechnology, vol. 5, no. 3, pp. 247–253, 2009.
23. Mukherjee, D. S. Ray, A. Thakur, and A. Basu, “Biocatalytic synthesis of metal nanoparticles and their applications,” Colloids and Surfaces B: Biointerfaces, vol. 82, no. 1, pp. 117–123, 2011.
24. L. Elechiguerra, J. L. Burt, J. R. Morones et al., “Interaction of silver nanoparticles with HIV-1,” Journal of Nanobiotechnology, vol. 3, no. 1, p. 6, 2005.
25. Ahmad, P. Mukherjee, D. Mandal et al., “Enzyme-mediated extracellular synthesis of CdS nanoparticles by the fungus, Fusarium oxysporum,” Journal of the American Chemical Society, vol. 124, no. 41, pp. 12108–12109, 2002.
26. W. Lee, S. W. Kim, T. Lee, and J. Y. Lee, “Effect of particle size and shape on the antibacterial properties of silver nanoparticles,” Journal of Physics: Conference Series, vol. 178, no. 1, Article ID 012037, 2009.
27. Rai, A. Yadav, and A. Gade, “Silver nanoparticles as a new generation of antimicrobials,” Biotechnology Advances, vol. 27, no. 1, pp. 76–83, 2009.
28. R. Jensen, M. D. Malinsky, C. L. Haynes, and R. P. Van Duyne, “Nanosphere lithography: tunable localized surface plasmon resonance spectra of silver nanoparticles,” The Journal of Physical Chemistry B, vol. 104, no. 45, pp. 10549–10556, 2000.
29. Zhang, X. Liu, W. G. Sun, and T. Xia, “Catalytic activity and thermal stability of silver nanoparticles synthesized via bio-reduction,” Materials Chemistry and Physics, vol. 105, no. 1, pp. 145–150, 2007.
30. Singh, N. Jain, and M. L. Singhal, “Microbial synthesis of metal nanoparticles: current trends and future prospects,” Critical Reviews in Biotechnology, vol. 29, no. 3, pp. 192–201, 2009.
31. Yang, H. Liu, Y. Li, and D. Du, “Biogenic synthesis of highly stable silver nanoparticles by the culture supernatant of Staphylococcus epidermidis,” Materials Letters, vol. 65, no. 8, pp. 1191–1193, 2011.
32. Pal, Y. K. Tak, and J. M. Song, “Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the Gram-negative bacterium, Escherichia coli,” Applied and Environmental Microbiology, vol. 73, no. 6, pp. 1712–1720, 2007.
33. Dobrucka and S. Długaszewska, “Antimicrobial activities of silver nanoparticles synthesized by using water extract of Polygonum hydropiper,” Advances in Natural Sciences: Nanoscience and Nanotechnology, vol. 6, no. 3, Article ID 035015, 2015.
34. Ahmad, H. K. Bhatt, and R. K. Wani, “Environmentally benign synthesis of nanoparticles using plant leaf extracts and their potential antimicrobial applications,” Nano Research Letters, vol. 8, no. 1, p. 473, 2013.
35. Bankar, B. Joshi, A. R. Kumar, and S. Zinjarde, “Banana peel extract mediated synthesis of gold nanoparticles,” Colloids and Surfaces B: Biointerfaces, vol. 80, no. 1, pp. 45–50, 2010.
36. A. Majumder, B. M. Mondal, and R. Ghosh, “Enhanced optical and catalytic properties of metal nanoparticles synthesized via plant extract,” Journal of Nanoscience and Nanotechnology, vol. 15, no. 7, pp. 5207–5215, 2015.