1 Introduction

The leading cause of human diseases, including most deaths each year, are food-borne infections [1]. Staphylococcal food poisoning, salmonellosis, listeriosis, hemorrhagic colitis, campylobacteriosis, and other serious food-borne illnesses are among the many health issues caused by consuming contaminated food [2, 3]. The genus Staphylococcus is a significant group of bacteria that cause serious food poisoning. It is a significant Gram-positive food-borne bacteria (GPB), facultatively anaerobic, and catalase-positive, found in cheese, milk, and meats as well as various environmental sources such as soil, water, and air [4]. Following the production of a variety of heat-stable enterotoxins, including type 1 toxic shock syndrome toxin (TSST-1), it is regarded as one of the most common causes of food intoxication. Staphylococcus spp. pose a significant threat to the food business because they continuously produce virulent factors such as the efflux pump, quorum sensing, biofilm, and different enzymes that include staphylokinase, coagulase and protease [5, 6]. For instance, S. aureus primarily produces enterotoxins that cause gastrointestinal distress, such as nausea, vomiting, and diarrhoea [4, 6]. Also, S. haemolyticus forms biofilms on surfaces used for food processing, tools, and packaging materials. These biofilms make the bacteria remain in food environments longer and cause contamination [5]. The enzymes, hemolysin and protease, can degrade host tissues and contribute to the survival of the bacterium in food products, potentially leading to spoilage and increasing the risk of foodborne illness. Hence, controlling Staphylococcus spp. is exceedingly challenging.

In animal husbandry, antibiotics are frequently used to treat illnesses. Animal health benefits by this approach and contributes to global food security. Antimicrobial resistance (AMR) has emerged and spread among food-borne pathogens because of the abuse of antibiotics, particularly in food producing animals [7, 8]. AMR has been listed as one of the top ten public health concerns to human life by the World Health Organization [9].

The rise of alternative approaches using nanoparticles (NPs) with plants and their products to combat the global threat of AMR has been forced by the increasing development of resistance to antimicrobials traditionally used in the management of animal infections, thus ensuring food safety and security [10]. Due to their observable characteristics including their high surface area to volume ratio, plant-derived nanoparticles (PDNPs) have proven to be a preferable choice in this regard. Their affinity for microbial membranes is improved by their huge surface area to volume ratio. Additionally, the positive charge on the NPs increases the affinity towards the negatively charged membrane of bacteria cells. Bacteria may not easily develop resistance to NPs because NPs simultaneously target a variety of bacterial death mechanisms [11]. Due to their distinctive physical and chemical characteristics, silver nanoparticles (AgNPs) are now utilized in creative ways in the field of nanomedicine [12]. Furthermore, they have a broader mode of action [13], low cytotoxicity [14] and are potent antibacterial agents [15].

Green synthesis is an easy and practical alternative to chemical and physical methods for the synthesis of metallic NPs, which involve organic solvents and other harmful substances [16]. In addition, it is economical and environmentally friendly. The use of biological agents such as bacteria, fungi, plants, and algae are a component of green synthesis techniques. However, plant extracts are being used more frequently because they are effectively reducing and capping agents, and because reducing the metal particles does not need intense cultivation time as compared to microbes [17]. Vachellia nilotica (L.) P.J.H. Hurter and Mabb subsp. Kraussiana (Benth.) Kyal. and Boatwr. (Fabaceae) aqueous extract was used in the present investigation for the synthesis of NPs. Elamary et al. [18] documented the existence of bioactive phytochemical components in the aqueous extract of V. nilotica and its capacity to effectively decrease biofilm activity of several Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. V. nilotica has also been documented to exhibit antimicrobial efficacy against Staphylococcus spp. strains that are resistant to conventional treatments. X-ray diffraction (XRD), followed by ultraviolet–visible spectrophotometry (UV/Vis), fourier transform infrared (FTIR), transmission electron microscopy (TEM), and scanning electron microscopy-energy dispersive X-ray (SEM–EDX), were some of the physical techniques used to characterize the bio-synthesized AgNPs. The MDR Staphylococcus spp. was used to test the antibacterial effectiveness of the green-synthesized AgNPs.

2 Materials and methods

2.1 Plant collection and preparation of plant extract

Fresh V. nilotica leaves were collected from Airport View (coordinates 25.8491°S, 25.5664°E), Mafikeng, North West Province, South Africa. The region is situated within the savanna biome, which is characterised by a combination of grasslands and dispersed trees. The climate is characterised as semi-arid to subtropical, featuring hot summers and mild, dry winters with annual precipitation ranging from 400 to 600 mm, predominantly during the summer months. The common tree species include Vachellia sp., Boscia albitrunca, and Sclerocarya birrea (Marula). The study area is predominantly defined by agricultural activities, specifically livestock farming and maize production. The voucher specimens were deposited at the S.D. Phalatse Herbarium (UNWH) located at the Department of Botany, Faculty of Natural and Agricultural Sciences (FNAS), North-West University, Mafikeng Campus, South Africa. A voucher number, JA91223, was issued following the positive identification of the plant. The samples were gently washed in distilled water to remove all debris and air-dried at room temperature. Following drying of samples, the leaves were ground to fine powder using an electric blender. A continuous heat extraction was set up using an improvised soxhlet extractor apparatus. This setup contained 100 mL of distilled water and 10 g of fine powder wrapped in filter paper. After extraction, the solution was filtered through a Whatman filter paper No.1 and the final solution was used as stock solution for further experimental use. The method adopted for plant extraction was as described by Alduraihem et al. [19].

2.2 Test bacterial strains

The bacterial culture used were environmental strains of multi-drug resistant (MDR) Staphylococcus haemolyticus with accession numbers JAVSMG000000000, JAVSMH000000000 and JAVSMI000000000 respectively [6]. A plethora of virulent and resistant genes revealed within the genomes of these strains indicate their potential public health implications, the severity of the illnesses they can cause in both animals and humans, and the difficulties related to their management.

2.3 Biosynthesis of silver nanoparticle

For the green synthesis of silver nanoparticles (AgNP), we used the aqueous leaf extract of V. nilotica as a reducing agent while silver nitrate (AgNO3) (Glentham Life Sciences GK7224, 99.8%) served as the precursor. For the synthesis, the standard method described by Hemlata et al. [20] was adopted. A mixture was prepared by combining 140 mL of an aqueous solution of AgNO3 with a concentration of 0.1 N and 70 mL of leaf extract, maintaining a ratio of 2:1. The resulting mixture was then subjected to continuous stirring while being heated at a temperature of 80 °C for a duration of 3 h. The transition from yellow to dark brown served as a preliminary indicator of AgNP formation. The green-synthesized NPs were separated from the mixture by centrifugation at 15,000 g for 20 min. This procedure was carried out three times to eliminate the free silver linked to AgNPs. The supernatant was discarded, while the pellet was kept in the microwave oven for exposure to heat and left until completely dried. The dried particulate matter obtained was hence referred to as the bio-synthesized AgNPs.

2.4 Characterization of the bio-synthesized silver nanoparticles

2.4.1 X-ray diffraction analysis

To determine the crystalline nature of AgNPs, an X-ray diffractometer (XRD) (Shimadzu, XRD-6000) equipped with a Cu Kα radiation source with a wavelength of 1.54 Å and Bragg angle of 30° ≤ 2θ ≤ 80, employing Ni as a filter, was used to investigate the X-ray diffraction of produced NP [21,22,23].

2.4.2 Ultraviolet–visible (UV/Vis) spectroscopy analysis

To quickly identify the synthesized AgNPs, UV/Vis spectroscopic examination was first carried out using spectro UV 2080 double beam [22]. By keeping track of the absorbance for the sample at wavelengths between 300 and 600 nm, the bio-reduction of Ag+ in an aqueous solution was monitored for completeness.

2.4.3 Transmission electron microscopy (TEM) and scanning electron microscope (SEM)

The average size distribution and surface morphology of the synthesized AgNP were determined using both transmission electron microscope (TEM) and scanning electron microscope (SEM) [22]. For three-dimensional imaging, scanning electron microscopy scans the materials using an electron beam. It is frequently utilized in studies on nanomedicine because of its great resolution. In transmission electron microscopy, a beam of electrons is transmitted through a tiny specimen to reveal the inside of the material. The images were analyzed using ImageJ software (1.6.0_12). These methods are regarded as some of the most effective ways to convey knowledge about NPs.

2.4.4 Fourier transform infra-red (FTIR)

To identify the potential biomolecules involved in the reduction and stabilization of AgNPs, FTIR spectra were scanned using an ALPHA interferometer (Alpha II Bruker Optik GmbH, 76275 Ettlingen, Germany) from 4000 to 500 cm−1 transmittance by KBr pellet method [23].

2.5 In vitro antibacterial assessment of synthesized AgNP

A microbial suspension was made from a single bacterial colony equivalent to 0.5 McFarland standards to assess the antibacterial activity using the disk diffusion method [24]. Separate cultures of the bacterial strains were cultivated on a Mueller–Hinton agar medium. On the surface of each plate, eight discs with varied concentrations of bio-synthesized AgNP (0.625, 1.25, 2.5, 5, 7 and 10 mg/mL), dimethyl sulfoxide (DMSO) used as a negative control, and a standard antibiotic; chloramphenicol (30 µg) used as positive control were laid out. Each sample was deposited in 20 µL portions onto each disk. After that, the plates were placed in a 37 °C incubator. The inhibitory zone was seen after 24 h. In each experiment, the inhibitory diameter zone was measured at least three times, and the mean was then computed.

2.5.1 Determination of the minimum inhibitory concentrations (MIC) of the particle

Using MIC, the inhibitory potential of the AgNPs product against the tested MDR bacterial strains was assessed. In accordance with the Clinical and Laboratory Standards Institute (CLSI) recommendations, the MIC was established using the broth microdilution method [25]. The two-fold microdilution method was used to perform MIC in 96-well microtiter plates. By diluting AgNPs in DMSO to the required commercial concentration, the solution was subsequently diluted by one-tenth in sterile Mueller Hinton Broth (MHB). Except for the negative control (drug-free) well, each well contained the antimicrobial agent (AgNP), the microbial inoculum at a concentration of 108, and the microbial growth medium (MHB). The plates were then incubated for 24 h at 35 ± 1 °C. The MIC is the level at which bacterial growth in microdilution wells was completely inhibited, which could be seen visually under transmitted light by the absence of turbidity. Additionally, each isolate MIC was calculated in triplicate to verify the results, and values were expressed as the average of three different experiments.

2.5.2 Determination of the minimum bactericidal concentrations (MBC) of the particle

Following broth microdilution, the MBC was identified by sub-culturing 50 µL from each well that had no discernible turbidity on MHA plates and incubating the plates at 35 ± 1 °C for 16–18 h. The MBC was established as the lowest concentration of NPs that totally suppresses bacterial growth after the plates were examined for the presence or absence of bacterial colonies. The MBC/MIC ratio was used to evaluate the NPs product's activity, with scores of 1, 2, and 4 deemed bactericidal and values of > 4.62 considered bacteriostatic. The CLSI [25] technique for antibacterial medicines was adopted in evaluating the NP’s activity as being either bactericidal or bacteriostatic.

2.6 Determination of the mechanism of action of the particle

In this investigation, tests for membrane damage, DNA leakage, and protein leakage were used to identify the antibacterial mode of action of the bio-synthesized AgNP.

2.6.1 Antibacterial activity of the bio-synthesized AgNP on cell membrane

This experiment was conducted following the protocol described by Ramalingam et al. [26] with modifications. After being exposed to NP at a bacteriostatic dose, the test pathogens’ cell membrane integrity was inspected under a microscope. The first step was to dispense 100 µL of the test agent to microtiter wells designated for the trials. The wells were then filled with 100 µL of the 0.5 McFarland standardized (108 CFU/mL) test isolates and incubated at 37 °C for 24 h. The suspension was then given 10 µL of trypan blue solution, which was then incubated for an additional hour at 37 °C [27, 28]. A light microscope was used to examine 10 µL of the final suspension. As a control, a test isolate not subjected to NP treatment was employed.

2.6.2 Antibacterial activity of the bio-synthesized AgNP on cell DNA

DNA damage (leakage) in the tested bacteria cell was examined using gel electrophoresis. A volume of 500 µL of sterile nutrient broth was carefully dispensed into each Eppendorf tube. Thereafter, 100 µL of bacteria (108 CFU/mL) was added and then incubated for 8 h at 37 °C. After that, the tube received 500 µL of the bactericidal concentration of the test agent (AgNP) and it was further incubated at 37 °C for an additional 16 h. Following 24 h of incubation, the suspension was centrifuged at 10,000 rpm for 5 min, and the supernatant was removed from the pellet. The pellet was filled with precisely 100 µL of sterile, distilled water before being vortexed. In addition, proteinase K was included to break DNA-associated proteins. Finally, DNA was eluted from the suspension. To verify the existence of DNA, the final DNA was seen in gel electrophoresis. Cells that had not been exposed to NP served as the control group.

2.6.3 Antibacterial activity of the bio-synthesized AgNP on cell protein

By using a slightly modified Biuret assay technique, the cellular protein leakage of the bacterial isolates grown with the sub-inhibitory concentration of the bio-synthesized AgNP was determined [29, 30]. An aliquot containing 500 µL of 24 h test isolate at 0.5 McFarland standard (108 CFU/mL) was introduced aseptically to an Eppendorf tube containing nutrient medium, and the tube was incubated at 37 °C for 8 h. After 16 h incubation at 37 °C, 500 µL of the synthesized AgNP (MIC) solution was introduced to the test tube. At the 24 h incubation stage, Proteinase K was also added to the spent medium to facilitate the breakdown of extracellular proteins. The suspension was centrifuged at 10000X for 5 min before the supernatant was discarded. To the pellet, exactly 100 µL of sterile distilled water was added, and the mixture was vortexed. After filtering out the cellular debris, DNase and RNase were added to the suspension to break down the DNA- and RNA-associated proteins. The suspension was supplemented with a 100 µL solution of copper sulfate and mixed after which drops of sodium hydroxide (NaOH) were introduced. The mixture was observed for colour change (bluish- violet colour indicates the presence of cellular protein).

3 Results

3.1 Biosynthesis of silver nanoparticles (AgNP)

In this study, a thorough investigation of the biosynthesis of AgNPs using extracts from natural plant, V. nilotica, was conducted and reported. V. nilotica natural plant extract was added to the solution of AgNO3, and the aqueous silver ions were then converted to AgNPs. After around 3 h of the reaction, it was noticed that the colour of the solution changed from yellow to bright yellow, then to dark brown, which indicated the synthesis of AgNP (Fig. 1).

Fig. 1
figure 1

Vachellia nilotica plant extract mixed with silver nitrate solution

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3.2 Characterized silver nanoparticles

3.2.1 X-ray diffraction analysis

The crystalline character of produced AgNPs is revealed by the XRD spectrum, which exhibits four intense peaks at 38.3°, 44.4°, 64.6°, and 77.6° of 2Ɵ of X-axis and 111, 200, 220, and 311 Bragg reflections of Y-axis, respectively (Fig. 2).

Fig. 2
figure 2

XRD of Vachellia nilotica leaf extract synthesized silver nanoparticle

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3.2.2 UV/Vis spectroscopy

The bio-reduction of Ag+ to Ag0 was observed using UV–vis spectroscopy. AgNPs made from aqueous extracts of V. nilotica displayed silver surface plasmon resonance at 456 nm in the absorption peak (Fig. 3) of the mixture of Ag nitrate solution and plant extract.

Fig. 3
figure 3

UV–Vis spectrometry of (A) Vachellia nilotica leaf aqueous extract and (B) silver nanoparticle stabilized in Vachellia nilotica leaf extract

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3.2.3 Scanning electron microscope (SEM) and transmission electron microscopy (TEM)

The SEM analysis depicts the particle size and morphological characteristics of the NP respectively as seen on the micrograph, which confirmed that the development of the NP looked to have tight forms and high surface diameters, demonstrating the aggregation at different magnifications (Fig. 4). The SEM image shows that the biosynthesized AgNPs are predominantly spherical, which is a typical morphology for AgNPs synthesized using green methods. The NPs exhibit polydispersity, showing a mixture of individual NPs and clusters, indicating some degree of aggregation. The spherical shape of AgNPs observed in SEM supports the XRD-determined FCC crystal structure, which favours isotropic growth.

Fig. 4
figure 4

Scanning Electron Microscope (SEM) image of the biosynthesized nanoparticle

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Figure 5A displays a TEM picture of the bio-produced AgNP and demonstrates increased particle size. The NPs exhibit mostly spherical shapes, with some having irregular, polygonal, or slightly faceted structures. Given the abundance of irregular AgNPs in the TEM picture, it was clear that Ag+ and V. nilotica leaf extract were the catalysts to produce the AgNPs. The larger AgNPs could represent the accumulation of smaller ones. Some NPs show a truncated triangular or hexagonal morphology, which is common for AgNPs synthesized under green synthesis. Some NPs exhibit lattice fringes, indicative of their crystalline nature. The TEM image was used to estimate the particle sizes of the AgNPs. An average particle size of 61 nm was obtained for the synthesized AgNPs as displayed in Fig. 5B.

Fig. 5
figure 5

A Transmission Electron Microscopy (TEM) images of biosynthesized silver nanoparticles (AgNPs), B Particle size distribution

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3.2.4 Fourier transform infra-red (FTIR)

The identification of several biomolecules that were adsorbed on the surface of AgNPs as well as their potential role in the modification and reduction of NPs were investigated in the FTIR range. The FTIR spectra display the vibrational bands of AgNPs alongside the aqueous leaf extract and AgNO₃ precursor, which are all related to each other (Fig. 6). The main broad and strong vibrational band at 3288 cm⁻1 is caused by the O–H stretching of the alcohols from the leaf extract. The peak at 2917 cm⁻1 is attributed to C–H stretching from organic stabilisers, whereas the peak at 1617 cm⁻1 is due to C=O stretching from carboxyl, aldehyde, or ketone groups associated with AgNPs capping. The signal observed at 1331 cm⁻1 serves as an indication of C=C, implying the presence of phenolic or flavonoid compounds resulting from green synthesis. A pronounced signal at 1020 cm⁻1 was detected due to C–O stretching, indicating the presence of organic molecules on the surfaces of AgNPs, whereas a peak at 825 cm⁻1 signifies Ag–O or Ag–N bonds arising from metal–ligand interactions with silver.

Fig. 6
figure 6

FTIR spectra for silver nitrate (AgNO3) precursor, Vachellia nilotica aqueous extract, and biosynthesized silver nanoparticle (AgNPs)

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3.3 Antibacterial effect of the bio-synthesized nanoparticle

In the current study, the antibacterial potency of the sample showed that each strain had varying levels of inhibition (Table 1). At 0.625 mg/mL, the AgNP had the lowest activity against the studied bacterial strains, and the zones of inhibition (ZOI) were 7.00 ± 0.57 mm. In contrast, at 10 mg/mL, ZOI was highest against isolate S. haemolyticus NWU MK-U2 with 30.07 ± 0.82 mm and against isolates S. haemolyticus NWU MK-S3 and S. haemolyticus NWU MK-U1 with readings of 28.00 ± 0.91 mm and 27.01 ± 0.83 mm, respectively. Zones of inhibition (ZOI) values were 25.04 ± 0.58 mm against isolates S. haemolyticus NWU MK-U2 and S. haemolyticus NWU MK-U1 and 27.00 ± 0.61 mm against isolate S. haemolyticus NWU MK-S3 using chloramphenicol (30 µg) as a positive control.

Table 1 Antibacterial efficacy of the bio-synthesized silver nanoparticle (AgNP) against the test isolates
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3.3.1 Minimum inhibitory and bactericidal concentrations

Table 2 displays the results of the MIC and MBC of the bio-synthesized AgNP on the examined bacterial strains. For all strains that were evaluated, the MIC and MBC of AgNPs were determined to be 2.5 and 5 mg/mL, respectively.

Table 2 Minimum inhibitory and bactericidal concentrations of the synthesized nanoparticle against the test isolates
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3.4 Mechanism of action

3.4.1 Effect of the bio-synthesized AgNP on the bacterial cell membrane

The damaging effect of the synthesized AgNP at the tested concentration on the tested bacterial strains was observed (Fig. 7). The neutralization of the cell membrane surface charge by exposure to the biosynthesized AgNP was observed to decrease membrane integrity and increase permeability. Viable cells do not retain the colour of the trypan blue solution while non-viable retained the colour.

Fig. 7
figure 7

Light microscopy of the microbial cells treated with the bio-synthesized silver nanoparticle and the untreated. A Control (untreated cells) show viable cells (colourless cells), B and C treated cells showing non-viable cells retaining colour of trypan blue

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3.4.2 Effect of the bio-synthesized AgNP on the cell DNA and protein

Following the visualization of gel electrophoresis, the DNA of the treated group was studied. Results from this experiment showed that no DNA band was observed from the treated group as opposed to the control cell. This experiment revealed the effect of the synthesized AgNP on the cellular protein leakage.

The DNA-associated protein was released following the modification of the cell membrane by the AgNP. This was confirmed by the colour change observed upon the addition of copper sulfate and drops of sodium hydroxide solutions to the extracted DNA.

4 Discussion

Saponin, tannin, alkaloid, glycosides, flavonoid, proteins, and phenols have reportedly detected in the aqueous extract of V. nilotica [31,32,33]. The activation, growth, and termination stages in the synthesis of plant-derived NPs are all distinct stages. Metal ions are reduced during the early step of activation, then grow as smaller NPs fuse together to form larger ones and finally reach their final size during the termination stage [34, 35]. As the reaction went along, the colour of the AgNO3 solution and plant extract mixture gradually altered. This colour shift results from the reduction of NPs caused by the phytochemicals found in the plant leaf extract. Biological agents are thought to function as reducers, stabilizers, or both during the production of NPs [36]. Polysaccharides, polyphenolic alkaloids, saponins, and terpenoids are examples of plant-produced phytochemicals that reduce metal ions or metal oxides into zero-valence metal NPs [37] and then maintain their stability [38]. Sheikh and Ishnava [39] reported that after 3 h of reaction, the mixture’s colour changed to brownish yellow, making reduction of AgNO3 clearly apparent. The length of the incubation period directly correlated with an increase in brown colour intensity. This may be due to the decrease of AgNO3 and activation of the surface plasmon resonance (SPR) action [40]. AgNPs produced in the medium have a SPR, which accounts for the colour change pattern and large peak observed in UV–vis spectrophotometry. These NPs absorb light at various wavelengths and are stimulated as a result of charge density at the conductor/insulator interface, each of which results in a distinct peak on UV–vis spectrophotometry [22].

By using UV–VIS spectroscopy, maximum wavelength (λ) obtained in this study was 456 nm, which offered strong support for the arrangement of AgNPs. Ahmed et al. [41] reported an absorbance peak in the range of 436–446 nm using Azadirachta indica aqueous leaf extract. According to several other research [19, 39, 42, 43], the highest reduction of AgNO3 into AgNPs occurs between 375 and 450 nm. Metal NPs have free electrons, which helps SPR comprehend the process since the metal NPs' electrons are connected in vibration with the light wave. Kumar et al. [22] revealed the AgNPs integrated from Adansonia digitata L. fruit pulp extract UV–VIS spectra at 434, 280, and 247 nm. Similarly, Alduraihem et al. [19] confirmed the biosynthesis of AgNP using V. nilotica pod extract with the SPR peak at 375 nm.

According to the observed peaks and the crystalline structure of the bio-synthesized AgNPs, which was confirmed by the analytical method of XRD, the major component of the synthesized NPs was silver. No additional prominent peaks indicative of contaminants was discovered in the XRD patterns. The face-centered cubic character of the synthesized AgNPs is indicated by the four intense peaks at 38.3°, 44.4°, 64.6°, and 77.6° of 2Ɵ of the X-axis, which correspond to 111, 200, 220, and 311 Bragg reflections of the Y-axis. Similar reports have been highlighted by a couple of studies [22, 44, 45].

The average particle size for the silver NP synthesis mediated by V. nilotica leaf extract in this study was 61 ± 0.29 nm, and it has also been shown that bigger AgNPs particles are produced because of NP aggregation during sample preparation. SEM with an energy dispersive spectroscopy (EDS) equipment showed improved resolution and a higher percentage of NPs present, allowing for accurate characterization and particle morphology. Size, shape, and aggregation pattern of NPs are revealed in higher resolution investigations with TEM examination. Due to the utilization of electron energies greater than 20 kV in SEM, it produces improved resolution [22]. The SEM and TEM findings from this investigation are consistent with those from earlier studies that utilized the same plant to synthesize AgNP [19, 39, 46] but at variance with the report of Skiba and Vorobyova [45] who used orange peel extract to synthesize AgNP. Further, Telrandhe et al. reported the morphology of AgNPs fabricated using Bombax ceiba thorn extract to be primarily cylindrical with a few being rod shaped and long. Moreover, a cubic structure of AgNP with a size range of 35–55 nm synthesized by biological technique has been reported [47].

The potential biomolecules responsible for the stabilization of the freshly produced AgNPs were determined by FTIR studies. FTIR is dependent on light absorbance in the electromagnetic, infrared area between 4000 cm−1 and 500 cm−1 [22]. Our synthetic NPs exhibit large peaks at 3288 cm−1 and 1020 cm−1 in this instance. Similar results have been published by several authors [39, 48,49,50]. Since a (NH) C=O group member within the cage of cyclic peptides is involved in maintaining the NPs, the peak at 1617 cm−1 is attributed to the (NH) C=O group’s binding to NPs [48, 51]. The conversion of Ag+ to Ag0 NPs is therefore greatly aided by peptides. The C–O–C and C–OH vibrations of the protein/polysaccharide in the V. nilotica extract are identified as the absorption band around 1020 cm−1 and 3288 cm−1, respectively, in AgNP [52, 53]. The vibrational bands at 1331 cm−1 and 2917 cm−1 are associated with bonds like –C = C (ring), –C–O, –C–O–C, and –C = C (chain) are formed from water-soluble compounds like flavonoid contained in the extract [48, 49].

This study indicates that the outstanding in vitro inhibitory effect of V. nilotica-AgNPs against susceptible MDR bacteria likely results from the swift oxidation activity and liberation of Ag + ions and/or the high surface charge of these particles. Diverse determinants, such as AgNPs’ size, morphology, stability, and concentration, can affect their antimicrobial activity [8, 54, 55]. Evidently, the presence of AgNPs exhibits reduced interference on the observed effect. At physiological level, the bacterial surface exhibits an overall negative charge because of the dissociation of an excessive quantity of carboxylic and other functional groups present in the bacterial membrane. Quite a few studies have indicated that the electrostatic attraction between bacterial cells carrying a negative charge and AgNPs possessing a positive charge plays a crucial role in the effectiveness of AgNPs [56,57,58]. This study corroborates those of Dawy et al. [59] and Liu et al. [60]. In their studies, the authors reported a more pronounced inhibitory impact of AgNPs against Gram-positive bacteria (S. aureus and Bacillus subtilis) compared to Gram-negative bacteria (Salmonella, Escherichia coli and Pseudomonas aeruginosa). However, some researchers reported otherwise [61, 62]. This discrepancy could be attributed to the potential protective effect of lipopolysaccharide (LPS) in Gram-negative bacteria [63].

A few studies have used MIC and/or MBC as methods to assess the efficacy of AgNPs against bacterial strains [64, 65]. The study conducted by Egger et al. [64] assessed the impact of AgNPs on a limited sample size of Gram-negative and Gram-positive bacterial strains [65]. There was a significant difference in the MIC and MBC between the two groups, indicating a higher vulnerability with respect to Gram-negative bacteria. The authors postulated that this phenomenon may be attributed to the distinctive characteristics of the bacterial cell wall. Gram-positive bacteria possess several layers of peptidoglycan, measuring approximately 30 nm, in contrast to the cell wall of Gram-negative bacteria, which measures around 3 nm. Another potential scenario involves the prevalence of teichoic and lipoteichoic acids in Gram-positive bacteria, which possess a significant negative charge. This negative charge has the potential to attract and bind free Ag+ ions.

There is evidence linking the antibacterial impact of bio-synthesized metal NPs with a leakage of intracellular proteins, DNA, and ions through a more permeable membrane [66,67,68]. This is made possible by the affinity of the negative electron charge on the microbial cells and the positive charge on the metal NP. In other words, electrostatic binding of metal NPs and mechanical membrane disruption lead to osmotic and cell membrane stress [69,70,71,72,73]. Results from this study showed that the integrity of the microbial cell membrane was compromised, likely due to the neutralization of the cell membrane surface charge. This was revealed by the microscopic view of the cells which showed that the cells treated with AgNP had many non-viable cells that picked up the colour of the trypan blue as against the control cells which had many viable cells and hence could not retain the colour of the stain. In their studies, Yuan et al. [74] and Wang et al. [75] reported the disruption in the cell membrane which ultimately led to the leakage of macromolecules.

Based on the prevailing knowledge and comprehension, one of the primary harmful mechanisms of nanosilvers involves the potential of the metal to interact with proteins [76,77,78,79]. Protein leakage brought about by the effect of AgNP on microbial cell membrane thus increasing membrane permeability has also been reported [80]. Results from this study conform to such previous outputs. Here, the presence of DNA-associated protein using Biuret’s assay was confirmed. Silver ions interact with macromolecules such as proteins and DNA inside the cells, causing damage to the cell wall, preventing cell development, and disrupting metabolic processes in bacteria. The interactions of the Ag ion with the cell prevent protein synthesis, reduce the permeability of the cell membrane, and ultimately cause cell death [81]. It is generally recognized that silver, even nanosilver, can influence amino acids and proteins [82].

Many experiments in which cells were exposed to NPs demonstrated DNA damage [83,84,85]. The high attraction of Ag+ to the numerous phosphates in DNA likely contributed to this damage, which included nuclear fragmentation or physical attachment of the AgNPs to the DNA [86, 87]. As demonstrated by McShan et al. [82], nanosilver interacts with DNA fibers in S. aureus. This was attributed to the damage to cell structures which is the primary manifestation of cellular reactions to nanosilver. Nanosilver clearly influences the metabolism of living cells by interacting with their macromolecular structures in a direct and active fashion. This was also corroborated by Banerjee and Das; Hou et al.; and Levard et al. [88,89,90].

Despite achieving the goals, it is important to highlight the limitations associated with this study. Among the restrictions are the make-up of the plant extracts. This depends on elements such as solvents used, extraction techniques, and growth conditions. Despite their sometimes-benign reputation, plant-based NPs have the potential to exert cytotoxic effect in human cells. This study did not specifically experiment on the cytotoxic potentials of the biosynthesized NPs.

5 Conclusion

The current investigation applied a method that is environmentally sustainable, devoid of toxicity, and economically viable to produce silver nanoparticles (AgNPs) using leaf extract derived from V. nilotica as a reducing agent. In this approach, naturally existing materials served as reducing agents, specifically biomolecules such as phenols and proteins found in plant extracts. This method offers a straightforward and alternative solution to intricate physical or chemical synthesis protocols. NPs with an average particle size of 61 nm, exhibiting mostly spherical shape and crystalline structure, were synthesized using V. nilotica leaf extract. The confirmation of this synthesis is achieved through the utilization of various analytical techniques, including XRD, UV–Vis, FTIR, SEM, and TEM. In this study, biosynthesized nanoparticles (Vc-AgNP) were tested in vitro and found to be effective against MDR S. haemolyticus strains at MIC and MBC levels of 2.5 and 5.0 mg/ml, respectively. This suggests that the biosynthesized AgNPs can be used in antimicrobial coatings, catalysis, biosensors, and nanomedicine due to their small size and high surface-to-volume ratio. Hence, a promising option as environmentally friendly antimicrobial agent.

Given the potential of the synthesized NP as a biocontrol agent, it is imperative to ascertain its toxicity profile prior to its application. Therefore, it is advisable to do investigations on the relevant concentration in living organisms, as in vitro findings cannot be extrapolated to in vivo conditions due to the influence of pharmacodynamics and pharmacokinetics features. Furthermore, the potential for utilizing plant-derived silver NPs in the medical domain is promising. This involves directing the NPs towards the analyze specific site of infection, hereby enabling targeted drug administration. Additionally, these NPs exhibit enhanced bioavailability and sustained drug release inside the targeted tissues, while also improving the stability of the NPs as a potential drug.