Introduction

In recent years, one of the most pressing issues in medicine is the emergence of multidrug-resistant pathogens. The lack of new and efficient antimicrobials has been linked to an increase in multidrug resistance. This has resulted in global initiatives to identify new and more effective antimicrobial drugs, as well as novel and effective medicine delivery and targeting systems1,2,3. The utilization of nanoparticles as innovative biomaterials to achieve this is receiving global interest. Nanoparticles become a necessary viable therapy alternative against drug-resistant microbes4,5. Different physical, chemical, and biological methods were used for the synthesis of nanoparticles, for example, silver nanoparticles Ag NPs due to their safe and antimicrobial properties. Currently, biological methods, particularly green synthesis, are favored over traditional physical and chemical approaches due to their eco-friendly nature, cost-effectiveness, and sustainability. This method not only reduces the use of hazardous chemicals but also enables a scalable and efficient preparation process, making it a promising alternative for large-scale applications6,7,8 .

Green synthesis methods using natural sources, such as extracts from plants, microorganisms, and enzymes, have become the preferred method to synthesize AgNPs9,10. Metal ions are reduced by metabolites due to their intrinsic reductive properties, while the resulting nanoparticles are further stabilized by these metabolites, ensuring their stability and uniformity. These metabolites include proteins, sugars, terpenoids, alkaloids, polyphenols, and phenolic acids11,12. A variety of pathogens, including bacteria, fungi, and viruses, have been shown to be effectively eliminated by Ag NPs. To enhance wound healing and prevent infections, Ag NPs can be added to antimicrobial topical treatments, medical device coatings, and wound dressings13,14,15.

Insects and their products are used for the green synthesis of Ag NPs with various applications. Honey bee was used in different studies and showed potential antibacterial activity against microbial pathogens16,17 .

Lateef et al.18 observed the potential antibacterial activity of Ag NP biosynthesis from paper wasp nest (Polistinae) extract against multidrug-resistant pathogens. Moreover, antibacterial activities were observed in the green synthesis of Ag NPs using paper wasp hydrolysate19.

Paper wasps of the genus Parapolybia (Hymenoptera: Vespidae: Polistinae) are distributed in the Middle East and East Asia. In this genus, two species are distributed only in the Middle East: Parapolybia escalerae and P. persica20. P. escalerae is distributed in Iran, Iraq, Turkey, and Pakistan21,22,23. Although little is known about the biology and behavior of this species, recent studies from Iran and Iraq introduce this species as a paper wasp of honey producers with local interest as a valuable food product and traditional medicine (Fig. 1)24. Therefore, in this study, the honey of the paper wasp (Parapolybia escalerae) for the green and eco-friendly synthesis of Ag NPs (H-Ag NPs). The as-synthesized nanoparticles were used against a series of Gram-positive and Gram-negative bacteria.

Fig. 1
figure 1

(A) Paper wasp (Parapolybia escalerae). (B) Paper wasp honey.

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Materials and methods

Chemical materials and honey harvesting

Silver nitrate (Ag(NO3)2) and sodium hydroxide were purchased from Merck, Germany. Honey was collected from nests of P. escalerae, which were harvested in September 2023 from Bamo-Khoshk Mountain (34.948735 N, 45.769756 E), Diyala Governorate, Kurdistan Region, Iraq, in the natural habitat of this species.

Bacterial strains used in this study

Three bacterial strains were employed to assess the antibacterial efficacy of the H-Ag NPs, including Staphylococcus aureus (S. aureus) ATCC 25922, methicillin-resistant Staphylococcus aureus (MRSA), and multidrug-resistant Acinetobacter baumannii. The MRSA strain of S. aureus and A. baumannii were isolated from swab specimens of burn wounds in the emergency burn and plastic surgery center in Sulaymaniyah. The preliminary identification of both clinical isolates was done using selective media, and then the isolates were identified using the Vitek 2 system. The Kirby-Bauer disc diffusion method was employed to evaluate the antibiotic susceptibility of isolated bacteria, and the results were interpreted according to the standard values established by the Clinical and Laboratory Standards Institute (CLSI)25,26. PCR amplification of the mecA gene was utilized to confirm the S. aureus MRSA strain isolate by employing the primers mecA (sense, 5-TCCAGATTACAACTTCACCAGG; antisense, 5-CCACTTCATATCTTGTAACG )27. The PCR procedure consisted of 30 cycles at 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 1 min, followed by a final extension of 10 min at 72 °C and a hold at 4 °C. In addition, carbapenem resistance of A. baumannii was confirmed by amplification of the blaOXA−51 gene using primers(5-TAATGCTTTGATCGGCCTTG-3 (forward)28, and, 5-TGGATTGCACTTCATCTTGG-3 (Reverse), using a PCR thermocycler (Techne, USA), the PCR was carried out with 45 cycles at 95 °C for 2 min, 60 °C for 34 s, and 72 °C for 30 s, with a final extension of 5 min at 72 °C and a 4 °C hold.

Biosynthesis of Honey-Based silver nanoparticles (H-Ag NPs)

Initially, the honey was separated from the paper combs by filtration using sterile Muslin Cloth. 20 g of the honey was dissolved in 80 mL of deionized water. 15 mL of the honey was mixed with a filter-sterilized 1 mM Ag(NO3)2 and stirred well for 1 min. To initiate the reduction of Ag+ ions, the mixture’s pH was adjusted to 8 using Sodium Hydroxide (NaOH). The progression of the reaction was monitored by observing a visible color change from colorless to a pale yellow and brown solution, indicating the formation of silver nanoparticles. UV-Vis spectrophotometry was utilized to verify the formation of silver nanoparticles by scanning the reaction mixture for absorbance across the 250–750 nm wavelength range. Upon completing the synthesis of H-Ag NPs, the solution was centrifuged at 10,000 rpm for 10 min at room temperature to separate the nanoparticles from residual honey. The solid fraction (pellet) obtained after centrifugation was collected, and subsequently, the nanoparticles were annealed at 450 °C for one hour. To further purify the silver nanoparticles, the powder was washed multiple times with distilled water to remove any remaining honey residues. Following the washing process, the resulting residue (H-Ag NPs) was dried in an oven at 75 °C for 12 h29,

Characterization of biosynthesized H-Ag NPs

FTIR spectroscopy was employed to analyze surface functional groups responsible for the reduction of Ag⁺ ions and the stabilization of H-Ag NPs by using a Thermo Scientific Nicolet iS10 FTIR spectrometer. The spectra were recorded over the wavelength range of 4000–400 cm⁻¹. The crystal study of biosynthesized H-Ag NPs was investigated using an X-ray diffractometer (XRD) (PAN Analytical Xpert Pro, Netherlands). To determine the crystalline structure of the H-Ag NPs, X-ray diffraction (XRD) analysis was conducted using Cu-Kα radiation (λ = 1.54 Å), the diffracted intensities of the samples were recorded in the 2θ range of 10º–80º with a step size of 0.1°and a scanning speed of 1 steps/second. The morphology and particle dispersion of powdered H-Ag NPs were investigated by scanning electron microscopy (SEM) (Quanta 4500, FEI). The purity of the prepared nanoparticles and confirmation of elemental silver were confirmed by Energy Dispersive X-ray Spectroscopy (EDX).

Antibacterial activity of H-Ag NPs

The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations of biosynthesized H-Ag NPs were investigated against methicillin-resistant S. aureus, multidrug-resistant A. baumannii, and S. aureus (ATCC 6538 ), using the standard broth dilution method26.

Bacterial strains were grown overnight in the nutrient broth medium at 37 °C with shaking (180 rpm). The bacterial inoculums were adjusted to a concentration of 106 CFU/mL. Then, 100 µL of each bacterial strain was transferred to 96-well microtiter plates. Subsequently, H-Ag NPs were prepared in a two-fold dilution series, for a final concentration range from 1.09 to 560 µg /mL, and 100 µL were added to the 96-well microtiter plates with the bacterial cells. Column 12 of the microtiter plate had the highest concentration of Ag NPs, whereas column 3 had the lowest. Column 2 was the positive control, which contained both the medium and bacterial inoculum, whereas column 1 was the negative control, which contained only the medium. Later, the 96-well microtiter plates were incubated at 37 °C for 24 h. Then, the bacterial cultures’ optical density at 600 nm (OD600) was measured using a microplate spectrophotometer (Biotech µQuant, USA). Following MIC measurement, aliquots of 5 µl from each microtiter plate well were spotted onto nutrient agar plates to perform the MBC test. All plates were then incubated for 18 h at 37 °C. The lowest concentration that showed no visible growth after sub-culturing was recorded as the MBC value30.

Statistical analysis

The Statistical Package for the Social Sciences (IBM, SPSS Statistics, and Version 25) software was used to statistically evaluate the data from the triplicate antibacterial tests. For analysis, One-way ANOVA was employed to assess the statistical significance. Duncan’s multiple range test was utilized to compare means; p ≤ 0.05 was considered a significant difference.

Results and discussion

Biosynthesis and characterization of H-Ag NPs

The opportunistic pathogens methicillin-resistant S. aureus and multidrug-resistant A. baumannii are primarily responsible for infections linked to hospital environments, which have higher rates of death and morbidity. In this study, biochemical and molecular techniques were used for the identification of these two clinically notable bacteria. The biofilm-forming ability of A. baumannii was confirmed through PCR amplification of the blaOXA−51 gene, which revealed biofilm formation and antibiotic resistance. Additionally, the amplified mecA gene in isolated S. aureus prevents β-lactam antibiotics from attacking transpeptidases involved in the synthesis of bacterial cell walls of S. aureus.

Today, various nanoparticles are considered an important alternative to antibiotic therapy. Silver nanoparticles have become one of the most investigated nanostructures and have attractive physicochemical properties; therefore, they play an important role in various biological applications4. Honey contains natural reducing agents such as fructose, glucose, and vitamin C. Additionally, bioactive compounds like flavonoids and proanthocyanidins contribute to the reduction of Ag⁺ ions into Ag NPs while also serving as capping agents to prevent aggregation and agglomeration. Notably, the hydroxyl groups in flavonoids exhibit a strong affinity for silver ions, enhancing their role as effective reducing agents16.

Reduction of Ag+ ions takes place with the addition of NaOH. The base facilitates the opening of the glucose ring by the abstraction of the proton of the sugar ring oxygen and the metal ions oxidize glucose to gluconic acid, also proteins and/or enzymes can also perform a role in oxidation recaption, resulting in a fast reduction of Ag+ ions to Ag0 and a large amount of very tiny nanoparticles, resulting in sharp and intense surface plasmon resonance (SPR) (Fig. 2)31. In addition, the formation of SPR is due to collective electron oscillation around the surface mode of the Ag NPs32. The UV-Vis absorption spectra of the reaction mixture have an absorption peak at 431 nm; this evidences the presence of surface plasmon resonance of the metallic H-Ag NPs (Fig. 3A). Additionally, a change in color of the reaction mixture (aqueous honey solution and AgNO3) from pale yellow to dark brown has widely been considered an indicator for the synthesis of nanoparticles. In contrast, no color change was observed in the control flasks. According to Matar et al. (2023), the average particle size of the silver nanoparticles made with Turkish honey was 14.3 and 14.7 nm, and they displayed absorbance at 443 and 456 nm33. It was evident that the UV absorbance and particle sizes of silver nanoparticles based on honey varied34.

Fig. 2
figure 2

Biosynthesis of silver nanoparticles using aqueous solution of Parapolybia escalerae local honey.

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The bioactive compounds of both honey and the biosynthesized silver nanoparticles (H-Ag NPs) were traced by the FTIR spectrophotometer (Fig. 3B). In addition, FTIR analysis was performed to determine which potential biomolecules were responsible for capping and effectively stabilizing H-Ag NPs made with honey. Totally, 7 peaks were obtained in the case of honey. The broad band at 3273.80 cm− 1 is assigned for O–H stretching vibration, indicating the presence of alcohol or phenol as a reducing agent. The weak band at 2931.91 cm⁻¹ is assigned to C-H stretching vibration, indicating the presence of alkane, carboxylic acids, and amino acids18. Since honey consists of flavonoids, proteins, and sugars, the 1644.80 cm⁻¹ peak is probably associated with the C = O stretching vibration. The bands at 1414.96, 1341.70, and 1256.94 cm − 1 mainly come from the deformation of the O–CH group and C–C–H in the carbohydrate structure29. The bands at 1024.23 and 992.6 cm − 1 correspond to the C–H groups, as well as C–O intense stretching vibrations in the carbohydrate structure. The bands at 925.11, 818.81, and 520.02 cm − 1 are the most intense stretching vibrations in the honey sample belonging to the C–H groups, as well as C–O in the carbohydrate structure35. FT-IR analysis of H-Ag NPs revealed the presence of various bands at 3278.11, 2920.42, 2851.47,1742.48, 1640.49, 1515.52, 1413.52, 994.06, 873.40, 777.15, 508.52, 453.94 cm− 1 due to the interaction of various functional groups of biomolecules in the honey solution, which are merely similar to those obtained by honey, indicating the presence of alcohol, alkane, organic acids, and nitro compounds36. The noticed difference is that peaks indicating alcohol, nitro compound, and alkene decreased in intensity, indicating the exploitation of these compounds in the reduction and capping of silver nanoparticles. The crystal structure of H-Ag NPs was characterized using powder XRD (Fig. 3C). The XRD pattern of Ag NPs shows distinct diffraction peaks corresponding to the face-centered cubic (FCC) crystalline structure of silver. The most prominent peaks are usually observed at 2θ values of approximately 38.1° (111), 44.3° (200), 64.5° (220), and 77.4° (311), which align with the standard reference pattern (JCPDS No. 04-0783) for metallic silver. Among these, the (111) plane at 38.1° is typically the most intense, indicating the preferred crystallographic orientation of H-Ag NPs. The Ag NPs produced using our eco-friendly process were nanocrystalline in nature due to the presence of structural peaks in XRD patterns and the average crystalline size of about 29.79 nm. The Debye-Scherrer equation was used to determine the average particle size of silver nanoparticles produced using the current green technique.

$$\:D=\frac{K\lambda\:\:}{\beta\:\:cos\:\theta\:}$$
(1)

where D is the average crystallite size of H-Ag NPs, λ = 0.1541 nm is the wavelength of the X-ray source, β is the full width at half maximum of (FWHM) of the peak in radians, K is the Scherrer constant with a value from 0.9 to 1, and θ is the Bragg angle37 .

The broadening of the peaks suggests the nanoscale nature of the particles. Additionally, any shifts or extra peaks in the XRD pattern might indicate lattice strain, defects, or the presence of impurities. In biosynthesized H-Ag NPs, organic capping agents from biological extracts can also introduce slight modifications in peak intensities. Previous studies also reported similar kinds of XDR spectra for the biosynthesis of Ag NPs38.

The scanning electron microscopy (SEM) image of synthesized Ag NPs is shown in (Figs. 3E and D). The SEM image shows individual Ag particles as well as several aggregates. The energy dispersive spectroscopy (EDX) was used to verify the existence of Ag elements. The greatest peak in the resulting nanoparticle EDX spectrum was observed at 3 keV, which is the typical absorption of metallic silver nanocrystalline. The presence of gold (Au) in the EDX spectrum is a result of the sample coating throughout SEM imaging (Fig. 3F). Our findings were consistent with previous literature studies39. The anticipated elements from honey-derived capping agents (C, O, N, etc.) are commonly found in green-synthesized Ag NPs, attributed to the presence of carbohydrates, amino acids, and other bioorganic compounds that function as reducing and stabilizing agents. Multiple investigations verified this, as evidenced by prior research16. In the current work, the elimination of C, O, and N peaks in our EDX analysis can be ascribed to the calcination of Ag samples, where the temperature was elevated to 450 °C for one hour. Transmission Electron Microscopy (TEM) was employed to analyze the morphological properties of the H-Ag NCs, as illustrated in (Fig. 3G). The images indicate that the Ag mostly displays a nanosphere form. The mean particle size, as ascertained by TEM histogram (Fig. 3H), is approximately 28–30 nm.

Fig. 3
figure 3

(A) UV–vis spectra of H-Ag NPs and aqueous honey solution; (B) FTIR spectra of H-Ag NPs and aqueous honey solution; (C) XRD pattern of H-Ag NPs; (D and E) are SEM images of H-Ag NPs; (F) EDX of the H-Ag NPs; (G and H) are TEM and Histogram images of H-Ag NPs.

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Antibacterial activity

Antibacterial properties of H-Ag NPs were evaluated using the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays, and the antibacterial activity of the synthesized H-Ag NPs was assessed at different concentrations. The minimum amount of an antimicrobial agent needed to eliminate 99.9% of the bacterial population is known as the MBC. The MIC, on the other hand, is the lowest concentration of an antimicrobial agent that, during the incubation time, prevents visible bacterial growth without necessarily killing bacteria. The results revealed that the biosynthesized nanoparticles have antibacterial activity against Gram-positive and Gram-negative microbial strains, even at low concentrations, and their activity is dose-dependent. It was revealed that the MIC and MBC of H-Ag NPs against S. aureus (ATCC 6538) and S. aureus MRSA strain are 17.5 µg/mL. In addition, these H-Ag NPs can inhibit the growth of Gram-negative strain A. baumannii at lower concentrations (8.5 µg/mL) (Fig. 4).

The biosynthesized H-Ag NPs often had a greater effect on Gram-negative bacteria than Gram-positive ones. According to our research, the lack of thick multilayer peptidoglycan in nanoparticles resulted in comparatively strong antibacterial action against Gram-negative bacteria. Gram-positive bacteria are generally characterized by a thick, multilayered peptidoglycan, which may help them maintain their cell structure and protect them from severe environmental factors. The thicker cell wall renders Gram-positive bacteria comparatively more resistant to silver nanoparticles40.

Although the exact mechanism of action of silver nanoparticles is not established, numerous studies have demonstrated that the silver ions released by Ag NPs interact with compounds in the cell wall that contain phosphorus and sulfur. This interaction interrupts the formation of the cell wall and, as a result, creates tiny holes that ions and other foreign substances can enter. The cell wall enlarges and ruptures due to lysis, and intracellular osmotic pressure rises41,42.

Since silver is toxic to several essential bacterial components, it has a broad-spectrum antibacterial action. It interferes with the functioning of enzyme systems, such as respiratory cytochromes, which are necessary for the electron transport chains that produce energy. Silver disrupts these mechanisms, impairing cellular respiration, which lowers energy availability and kills bacteria43. Additionally, silver inhibits transcription and division by targeting microbial DNA and RNA. By interfering with the production of essential proteins and inhibiting replication, this interference successfully stops the growth of bacteria. These many processes demonstrate silver’s effectiveness in inhibiting bacterial growth and its possible application in treating antibiotic-resistant infections44.

Table 1 shows previous studies examining biosynthesized nanoparticles and their antibacterial activity, in contrast to our samples. The activities shown by the H-Ag NPs are consistent with several reports on the antibacterial activities of Ag NPs. For instance, new Ag NPs stabilized by bee honey, which had an average size distribution of 10–12 nm, demonstrated antibacterial activity against a variety of bacterial and fungal strains. The lowest MIC value against S.aureus was 2.81 mg/L, whereas Ag NPs demonstrated MIC values of approximately 5.62 mg/L for Salmonella typhi, Streptococcus mutans, Escherichia coli, and Candida albicans, respectively45.

Research by Lateef et al. (2016) reported biosynthesis of Ag NPs using paper wasp‘s hydrolysate as a reducing agent, which demonstrated potent antibacterial activity against three multidrug-resistant strains of Pseudomonas aeruginosa and Klebsiella granulomatis18. Similarly, another study conducted by de Souza et al. (2024) found that Ag NPs synthesized from honey bees exhibited bacteriostatic and bactericidal activity against E.coli and S. aureus. Thus, employing different honeys to create Ag-NPs in a green way can be an efficient and inexpensive method to combat bacterial multidrug resistance. Since honey-mediated silver nanoparticles are environmentally benign, have potential uses in biomedicine and the environment, and meet the growing need for sustainable nanotechnology, their prospects for green synthesis and characterization are promising35.

Fig. 4
figure 4

(A and B) Antibacterial activity of H-Ag NPs against multidrug-resistant A. baumannii, S. aureus (ATCC 6538), and S. aureus MRSA illustrated the MIC and MBC of nanoparticles in µg/mL (OD: optical density).

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Table 1 Comparison of the antibacterial activity of the green-synthesized H-Ag nps.using the aqueous honey solution with biosynthesized ag nps.
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Conclusions

This study underscores the innovative utilization of P. escalerae (paper wasp) honey as a natural, eco-friendly reducing and stabilizing agent, successfully facilitating the green synthesis of silver nanoparticles (H-Ag NPs). This approach not only advances sustainable nanomaterial production but also highlights the untapped potential of biologically derived resources in nanotechnology. The successful synthesis of H-Ag NPs was confirmed via UV–Vis spectroscopy, FT-IR, XRD, and SEM analyses, providing detailed insights into their optical, structural, chemical, and morphological properties. The findings revealed that H-Ag NPs offer a superior and more rapid alternative to conventional chemical synthesis methods. Remarkably, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against S. aureus (ATCC 6538) and methicillin-resistant S. aureus (MRSA) were determined to be 17.5 µg/mL. Furthermore, even at a lower concentration of 8.5 µg/mL, the nanoparticles effectively suppressed the growth of the Gram-negative pathogen A. baumannii, highlighting their broad-spectrum antibacterial potential. More research has to be conducted to evaluate the cytotoxicity of those NPs and test them against various pathogens. The full potential of this innovative strategy and its integration into multiple industries and scientific fields will be unlocked with further study and development in this field.