Synergistic antibacterial activity of biogenic AgNPs with antibiotics against multidrug resistant bacterial strains

2.1 Preparation of Fenugreek seed extract

The seeds of Fenugreek were procured from a Riyadh, Saudi Arabian market, and the Herbarium of King Saud University's College of Science verified their authenticity. After being washed with tap water, the seeds were rinsed three times with distilled water for a complete cleaning procedure. They were then left to air dry. To make a fine powder out of the Fenugreek seeds, a mechanical mortar was used. After that, 50 g of powder were dissolved in 200 ml of distilled water, and the mixture was then heated in flasks at 50 °C for half an hour. The subsequent mixture was agitated with a magnetic stirrer for 24 h at 25 °C. To get a clear filtrate, the Fenugreek seeds extract was next filtered using Whatman filter paper (1). The extract was sterilized by passing it through a 0.45  µm Millipore membrane filter, and then it was stored in the fridge at 4 °C until later use.

2.2 Green synthesis of AgNPs

Initially, 10 mL of the water extract obtained from Fenugreek seeds was added to 90 mL of a 1 mM solution of colorless AgNO₃ (Sigma-Aldrich, Missouri, USA). Subsequently, 90 mL of silver nitrate solution was mixed with 2, 4, 6, 8, and 10 mL of Fenugreek seed extract, each at a silver nitrate concentration of 1 mM. Additionally, different concentrations of AgNO₃ (0.2, 0.4, 0.6, 0.8, and 1.0 mM) were used to synthesize silver nanoparticles while maintaining a constant extract concentration (2 mL).

Subsequently, the reaction mixture underwent incubation at room temperature in a shaking incubator under dark conditions. The biosynthesis process of AgNPs was visually monitored, observing a color shift from colorless to a light brown hue. AgNPs were separated from the reduced mixture through centrifugation at 10,000 rpm for 10 min. The collected AgNPs underwent a thorough washing process with deionized water, repeated three times to eliminate impurities. Subsequently, the extract underwent sterilization through filtration, employing a Millipore membrane filter with a pore size of 0.45  µm. Finally, the purified AgNPs were dried at 80℃ in an oven for subsequent physicochemical characterizations (Yassin et al., 2022b, 2022c).

2.3 Physicochemical properties of the biogenic AgNPs

The physicochemical features of the biogenically synthesized AgNPs were explored through diverse methodologies as UV–Vis, TEM, EDX, FTIR, XRD and zeta potential analysis of the biosynthesized AgNPs.

2.4 Screening of antimicrobial efficacy of AgNPs against bacterial pathogens

The American Type Culture Collection provided four bacterial strains: A. baumannii (ATCC 43498), K. pneumoniae (ATCC 700603), E. coli (ATCC 25922), and P. aeruginosa (ATCC 9027). The selection of bacterial pathogens for antimicrobial investigations was based on their substantial impact on worldwide mortality and morbidity, which is mostly caused by their extensive antibiotic resistance. The antibacterial efficacy of biogenic AgNPs was examined against the identified bacterial strains using the disc diffusion technique. The bacterial colonies were collected, mixed with a saline solution, and adjusted to a concentration of 10⁸ cfu/mL using the 0.5 McFarland standard. After filling sterile Petri plates with sterile Mueller Hinton agar (MHA) media and inoculating them with the bacterial solution, 8 mm filter paper discs impregnated with 100 and 200 µg of AgNPs were added. Filter paper discs soaked with methanol solvent functioned as the negative controls, and Colistin sulfate as the positive control. Plates were incubated at 37 °C for 24 h after being refrigerated for 2 h to facilitate the diffusion of nanoparticles. A Vernier caliper was used to measure the inhibitory zone diameters. The broth microdilution test employing 96-well microtiter plates was used to evaluate the minimum inhibitory concentration (MIC) of AgNPs from Fenugreek seeds against the most sensitive strain of E. coli. In addition, the minimum bactericidal concentration (MBC) was determined by inoculating samples from MIC wells onto MHA plates. The MBC was defined as the lowest concentration at which no bacterial growth was observed.

2.5 Determination of synergistic potency of biogenic AgNPs with antibiotics

The combined efficacy of antibiotic-AgNPs against certain bacterial pathogens was evaluated via the use of disc diffusion assay. Fresh MHA plates were made and then inoculated with 0.5 mL of the bacterial solution that was prepared earlier. A separate set of discs was impregnated with both of colistin (10 μg) or norfloxacin (10 μg) and AgNPs (100 μg/disk) to examine their synergistic effect. The positive control discs were loaded with colistin (10 μg) and norfloxacin (10 μg) only, whereas the negative control discs were filled with filter paper discs soaked in methanol solvent. The moistened discs were allowed to dry before being positioned on the MHA plates that had been previously inoculated. Following that, the dishes were subjected to incubation at a temperature of 37 ℃ for a period of 24 h. The calculation of the increase in fold of inhibition area (IFA) was performed utilizing the subsequent equation:

(IFA) = (B² − A²)/A², where A was the diameter of the inhibition zone for colistin or norfloxacin antibiotics and B was the diameter of the inhibition zone for antibiotics and biogenic AgNPs combined together (Al-Otibi et al., 2023).

2.6 Determination of morphological cell deformations

Scanning electron microscopy (SEM) was utilized to assess the morphological changes in bacterial cells treated with biosynthesized AgNPs. The samples were processed for investigation as mentioned in a prior study (Almaary et al., 2023).

3.1 Optimization of the reaction conditions

Various volumes of the plant extract and varying concentrations of AgNO₃ were examined to determine the factors that contribute to achieving the highest yield of AgNPs. In this context, it was seen that the highest amount of AgNPs was produced when using a concentration of 1 mM of AgNO₃ and 10 ml of the plant extract. This resulted in the development of AgNPs precipitates with concentrations of 0.57 ± 0.04 and 1.47 ± 0.09 mg/ml, respectively (Fig. 1).

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Fig. 1

Effect of different concentrations of AgNO₃ and plant extract on the yield of AgNPs.

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3.2 Green synthesis of AgNPs

Fig. 2 showed the green fabrication of AgNPs using seed extract of Fenugreek. The colorless solution of silver nitrate (Fig. 2a) undergoes reduction when mixed with the liquid extract of Fenugreek seeds, leading to the creation of silver nanoparticles. The Fenugreek seeds include a variety of natural chemical components, including amino acids, flavonoids, saponins, alkaloids, and steroids. These components are responsible for the extensive usage of seed extracts in the environmentally friendly production of various metal and metal oxide nanoparticles (Jeevanandam et al., 2022). Furthermore, the extract phytochemicals function as capping and stabilizing agents, playing an important role in the production of AgNPs (Restrepo and Villa, 2021).

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Fig. 2

a) Green synthesis of AgNPs using T. foenum-graecum seeds extract, b) UV spectrum of Fenugreek seeds extract and their corresponding AgNPs.

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3.3 UV analysis of biogenic AgNPs and Fenugreek seed extract

The surface plasmon resonance (SPR) of the biosynthesized AgNPs was determined by analysing the Fenugreek seed extract and AgNPs with UV analysis. The coordinated motion of electrons within the conduction band of nanoparticles upon absorption of an electromagnetic wave is referred to as surface plasmon resonance. In this setting, Fenugreek extract shown the existence of two absorption bands at 251 and 507 nm. Consistent with the outcomes of a prior study, our results confirmed the presence of a distinctive peak at 500 nm in the UV spectrum of the water extract of red currant (Rizwana et al., 2022). The absorption behavior seen in Fig. 2b is caused by SPR of AgNPs, which occurs when conduction band electrons of the AgNPs undergo coherent oscillation in response to the electromagnetic field. This explains the presence of a broad band with distinctive peaks at 476 and 509 nm in the UV spectrum of plant extract, which were shifted to a higher wavelength in the UV spectrum of AgNPs at 480 and 519 nm. The results of our study were consistent with those of a prior publication (Syafiuddin et al., 2017a).

3.4 TEM analysis of AgNPs

TEM micrographs of AgNPs showed that the nanoparticles were spherical in shape with average particle size of 29.75 nm (Fig. 3a). The histogram of the particle size distribution indicated that the size of the AgNPs varied between 10 and 70 nm (Fig. 3b). The findings of our study aligned with a previous publication that showed the successful production of silver nanoparticles (AgNPs) using olive leaf extract. The report reported that the average particle size of the AgNPs was around 30 nm (Khalil et al., 2014). Additionally, another research used transmission electron microscopy (TEM) to analyze AgNPs morphology made from an aqueous leaf extract of Azadirachta indica. According to the research, these AgNPs had an average size of 34 nm (Ahmed et al., 2016). The measured particle size was smaller than in earlier publications, showing the effectiveness of the present approach for synthesizing AgNPs using the seed extract of T. foenum-graecum.

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Fig. 3

A) tem images of agnps, b). particle size distribution of agnps.

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3.5 EDX analysis of the biogenic AgNPs

EDX analysis was used to conduct elemental mapping of the biogenic AgNPs (Fig. 4a). The study revealed that the proportion of elemental silver was 73.42 %, whereas the percentage of oxygen was 26.58 %. The strong signals at 2.983 keV were attributed to silver atoms, whereas the signal at 0.525 keV was allocated to oxygen atoms. The mass percentage of elemental Ag was more than that reported in a recent work on the green synthesis of AgNPs using the endophytic fungus Talaromyces purpureogenus, which was isolated from Taxus baccata Linn. The relative mass percentage was found to be 67.26 % (Sharma et al., 2022). Additionally, the EDX examination revealed that the AgNPs synthesized utilizing leaf extract of Holoptelea integrifolia had an elemental content of 71.32 % Ag (Kumar et al., 2019).

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Fig. 4

A) edx spectrum of agnps (x axis refereed to the energy level (kev) whereas y axis refer to the counts), b) FTIR spectrum of Fenugreek seeds extract and AgNPs.

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3.6 FTIR analysis of the biogenic AgNPs

FTIR investigation of Fenugreek seed extract and AgNPs was done to detect the key functional groups contributing to the reduction, capping and stabilizing of AgNPs. In this setting, FTIR spectrum of the seed extract of Fenugreek displayed the existence of six peaks at 3395.87, 2081.10, 1637.88, 1386.34, 1115.38, and 662.29 cm⁻¹ (Fig. 4b). On the other hand, the FTIR spectrum of AgNPs displayed the existence of nine absorption bands at wavenumbers of 3416.42, 2926.18, 2370.75, 2047.32, 1628.06, 1388.77, 1114.91, 619.97 and 477.98 cm⁻¹. The wide spectral band observed at 3395.87 cm⁻¹ in the Fenugreek spectrum was identified as the result of O–H stretching of phenolic biomolecules. In AgNPs spectrum, this peak was shifted to a higher wavenumber at 3416.42 cm⁻¹, indicating that the extract phenols were adsorbed over nanoparticles surface (Hajebi et al., 2019). The spectra of AgNPs displayed two weak bands with wavenumbers of 2926.18 and 2370.75 cm⁻¹. These peaks were identified as the result of stretching vibrations of C–H and N–H bonds in alkanes and amines, respectively (Oves et al., 2022). In contrast, the peak seen at 2081.10 cm⁻¹ in the seed extract was shifted to a lower wavelength at 2047.32 cm⁻¹, these bands were attributed to the C=S stretching of sulfur compounds (Mistry et al., 2021). Moreover, the absorption band seen at 1637.88 cm⁻¹ in spectra of Fenugreek seed extract was shifted to a lower wavenumber at 1628.06 cm⁻¹, corresponding to the amide I bands of proteins (Das et al., 2013). The peak seen at 1388.77 cm⁻¹ in the spectra of AgNPs was displaced to a lower wavenumber of 1386.34 cm⁻¹, indicating the C–H bending of alkanes. Nevertheless, the peak detected at 1114.91 cm⁻¹ in the Fenugreek seed extract spectra was displaced to a higher frequency at 1115.38 cm⁻¹ in the AgNPs spectra, indicating the C-N stretching of amines (Deivanathan and Prakash, 2022). The strong peak detected at 662.29 cm⁻¹ was shifted to a lower wavenumber at 619.97 cm⁻¹ corresponding to S-S stretching (Raghava et al., 2021).

3.7 XRD analysis of AgNPs

XRD investigation was performed to affirm the crystalline structure of the eco-friendly synthesized AgNPs. XRD analysis showcased the existence of six diffraction peaks at 2 theta angles of 21.19, 32.28, 37.31, 49.34, 57.48 and 77.55° corresponding to the orientation planes of (1 0 0), (1 1 0), (1 1 1), (2 0 0), (2 2 0) and (3 1 1), respectively (Fig. 5). The XRD outcomes were in line with that of previous reports (Varghese et al., 2019). The detected peaks validate the polycrystalline characteristic of the synthesized AgNPs and may be ascribed to the typical JCPDS data of crystalline silver with a face-centered cubic (fcc) lattice structure, identified by the code COD 2300113. The additional peaks seen in XRD pattern correspond to the organic stabilizers that inhibit the aggregation of AgNPs (Jalilian et al., 2020). The bioprepared AgNPs were found to have a crystalline size of 28.71 nm, which was determined using Scherrer's equation.

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Fig. 5

XRD spectrum of AgNPs.

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The formula for calculating the crystalline size is given by D=0.9λ/ β cos θ, where λ represents the X-ray wavelength (1.54178 Å), k represents Scherrer's constant (k = 0.94), θ represents the diffraction angle (32.31°), and β represents the full width at half maximum (FWHM) of the most intense diffraction peak (0.301) (Yassin et al., 2023). The physicochemical features of the biogenic AgNPs were compared with that of previous literature as seen in Table 1.

3.8 Zeta potential analysis

Zeta potential analysis was done to estimate the zeta potential value of the eco-friendly synthesized AgNPs. The presence of high negative values suggests that anionic stabilizing agents are effectively coordinating with the AgNPs, resulting in a stable colloidal solution. This stability is achieved by the electrostatic repulsion between the negatively charged particles. The presence of negatively charged surfaces on AgNPs serves to inhibit the aggregation of nanoparticles and exert control over their shapes and sizes (Paosen et al., 2017). In this context, zeta potential value of AgNPs was detected to be –22.4 mV (Fig. 6a). However, the results from DLS displayed that the mean hydrodynamic nanosize of AgNPs was 166.9 nm (Fig. 6b). This size is larger than what was observed using TEM and XRD techniques. The difference might be accredited to the existence of capping biomolecules and the fact that DLS analysis measures both the nanoparticles diameter and the surrounding hydrate layers. The polydispersity index (PDI) of the biogenic AgNPs was estimated to be 0.364, which in agreement with the findings of a prior investigation that reported a PDI value of 0.365 for AgNPs synthesized using Ginkgo biloba leaf extract (Wang et al., 2019). This indicates that the synthesized AgNPs were effectively dispersed in water.

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Fig. 6

A) zeta potential value of agnps, b) DLS spectrum of AgNPs.

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3.9 Antibacterial efficacy of AgNPs against the concerned microbial strains

The antibacterial efficacy of AgNPs was detected against A. baumannii, E. coli, K. pneumoniae, and MRSA pathogens using a disc diffusion assay. E. coli strain showed the highest antibacterial susceptibility to the biogenic AgNPs at the two tested concentrations of AgNPs (50 and 100 µg/disc), demonstrating relative suppressive zones of 18.43 ± 0.35 and 21.32 ± 0.19 mm, respectively (Table 2). Nevertheless, the MRSA strain exhibited the least sensitivity to the biogenic AgNPs at the tested doses, resulting in inhibitory zones of 10.24 ± 0.21 and 11.26 ± 0.51 mm, respectively. The study findings demonstrated that AgNPs exhibit greater antibacterial efficacy against Gram-negative bacteria compared to Gram-positive bacteria due to the presence of a peptidoglycan layer in the cell wall of Gram-positive bacteria (Ramalingam et al., 2016). This layer, consisting of glycan strands cross-linked by short peptides and anionic glycopolymers known as teichoic acid, acts as a natural barrier that hinders the penetration of nanoparticles. In contrast, Gram-negative bacteria possess a cell wall that is thinner and has a lower amount of peptidoglycan. Although lipopolysaccharides (LPS), a crucial element of the Gram-negative bacterial cell wall, greatly enhances the strength and integrity of the membrane, its negative charge promotes the attachment of nanoparticles (Tavares et al., 2020). Previous research has shown that reactive oxygen species (ROS) play a significant role in the antibacterial action of biosynthesized AgNPs, as shown in an evaluation of AgNPs' ROS-mediated antibacterial activity against multidrug-resistant E. coli and S. aureus. Consequently, the elevated amount of ROS caused the bacterial membranes to be disrupted by increasing their permeability. This ultimately led to the disruption of the electron transport chain and the leaking of cellular content, leading to the bacterial cell death (Das et al., 2017). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were detected for E. coli strain which showed the highest sensitivity to AgNPs. The detected MIC and MBC values were found to be 50 and 100 µg/ml, respectively. The antimicrobial activity of AgNPs could be assigned to the capping agents adsorbed over the biogenic AgNPs as phenols, alkanes, amines, amide I bands of proteins, and alkanes as detected by FTIR analysis and this was in accordance with previous report (Roy et al., 2019).

3.10 Synergistic antibacterial efficiency of AgNPs with antibiotics

The AgNPs exposed the uppermost synergistic efficacy with colistin against A. baumannii strain with relative IFA of 0.53 whereas AgNPs displayed the highest synergistic proficiency with norfloxacin against P. aeruginosa with relative IFA value of 0.35 (Table 3). Our outcomes were in line with that of a prior investigation which reported the antimicrobial action of silver nanoparticles conjugated with colistin against E. coli, K. pneumoniae, and P. aeruginosa (Muenraya et al., 2022). The combined action of AgNPs and colistin antibiotic against the gram negative bacterial strains is due to the impact of colistin antibiotic on the lipopolysaccharide, a key component of the outer membrane. This leads to the disruption of the outer membrane, allowing AgNPs to enter the bacterial cells. AgNPs then exert their killing effect by producing reactive oxygen species, that oxidize the bacterial DNA, proteins and lipids, ultimately resulting in the demise of the bacterial cells (Tripathi and Goshisht, 2022). An earlier work documented the synergistic effect of norfloxacin and gallotannin-capped iron oxide nanoparticles, showing an increase in inhibitory zones from 13 mm to 17 mm and 19 mm against the two strains, respectively (Ahmed et al., 2021). The combined effect of AgNPs and norfloxacin might be accredited to AgNPs accumulation on the outer membrane of bacterial cells, triggering their disruption. On the other hand, norfloxacin antibiotic inhibits bacterial growth by interacting with bacterial topoisomerase IV and DNA gyrase, thereby preventing DNA replication (Huang et al., 2024).

3.11 Detection of bacterial cell deformations using SEM analysis

The morphological deformations of E. coli cells were investigated using SEM analysis. The control cells of E. coli were found to be normal, rod-shaped, and with structural integrity without any signs of deformation. Moreover, it was observed that E. coli cells subjected to AgNPs treatment exhibited aberrant morphological characteristics, including cellular damage, wrinkling, malformation, as well as a cracked outer surface and distorted cell membranes (Fig. 7).

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Fig. 7

Morphological deformations of E. coli cells exposed to biogenic AgNPs (the morphological deformations were pointed out by arrows).

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