Trachyspermum copticum aqueous seed extract-derived silver nanoparticles: Exploration of their structural characterization and comparative antibacterial performance against gram-positive and gram-negative bacteria

2.5.1 Well diffusion assay

According to the method described previously [19], after the preparation of the bacterial inoculums aseptically the cell density was modified to 0.5 McFarland standard (10⁸ CFU·mL⁻¹) coupled with covering the Mueller Hinton Agar (MHA) in the plates with the inoculums. After dispersing the AgNPs in deionized water at the concentration of 1 mg·mL⁻¹, 50 and 100 μL of the stock were separately poured into the wells with a diameter of 6 mm created in the contents of the plates. Moreover, 100 μL per well of 0.5 mM aqueous AgNO₃ solution and 100 μL per well of the aqueous extract of T. capticum (0.1% stock) were considered for comparison. Eventually, the plates were incubated for 1 day at 37°C.

2.5.2 Broth microdilution assay

2.5.3 Reactive oxygen species (ROS) assay

To evaluate the amount of ROS, known as unstable molecules damaging the biological compositions in cells, the TPR-ROS Assay Kit (Teb Pazhouhan Razi, Iran) was employed using 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) which is a ROS-sensitive probe. This fluorometric assay involved the hydrolysis of DCFH-DA to produce a non-fluorescent molecule (DCFH). This hydrolysis is facilitated by an ester hydrolysis enzyme. Subsequently, DCFH is rapidly oxidized to form strong green fluorescence dichlorofluorescein. The intensity of this fluorescence at λ _ex = 485 and λ _em = 535 is directly proportional to the amount of ROS present in the cells. For this study, bacterial strains were seeded in a 96-well plate at a density of 1.5 × 10⁸ cells per well and incubated for 24 h. The culture medium was then replaced, and the cells were treated with biofabricated AgNPs for an additional day. Following the treatment, the TPR kit protocol was followed, and the fluorescence intensity was measured using a Hybrid Multimode Reader Cytation 3 (BioTek Company, USA).

2.5.4 Flow cytometry-based quantitative assessment of cell viability

Initially, different concentrations of biofabricated AgNPs were added to reference strains of S. aureus (ATCC 25923), S. pyogenes (ATCC 19615), E. coli (ATCC 25922), and K. pneumoniae (ATCC 10031) at the concentration of 0.5 McFarland (10⁸ CFU·mL⁻¹) in the Sabouraud Dextrose Broth (SDB) medium. After incubating the components at 28°C and 125 rpm for 24 h, the samples were centrifuged at 8,000 rpm for 1 min followed by separating and discarding the supernatant. Then, the phosphate-buffered saline buffer was used to wash the plates. This step was repeated one more time and after adding 2 µg·mL⁻¹ of propidium iodide dye, the plates were incubated for 20 min. Finally, a flow cytometer (BD FACSCalibur™, USA) and FlowJo software (Tree Star, Inc., Ashland, OR, USA) were employed to assess the samples and analyze the data, respectively.

3.2.1 Well diffusion assay

Figure 6 shows the results of the well diffusion assay of AgNPs, aqueous silver nitrate solution, and T. copticum aqueous seed extract against all tested bacteria. The inhibition zone (IZ) of 50 and 100 µL of AgNPs with a concentration of 1 mg·mL⁻¹ and 100 µL of 1 mM AgNO₃ solution was found to be 23 ± 1, 23.33 ± 0.58, and 18.33 ± 0.58 mm, respectively against S. aureus (ATCC 25923). Notably, no IZ was found for 100 µL of 0.1% aqueous seed extract of T. copticum against S. aureus. Besides, the IZ of 50 and 100 µL of 1 mg·mL⁻¹ AgNPs and 100 µL of 1 mM AgNO₃ solution was found to be 24.33 ± 0.58, 24.33 ± 0.58, and 15 ± 1 mm, respectively, against S. pyogenes (ATCC 19615). Notably, no IZ was found for 100 µL of 0.1% aqueous seed extract of T. copticum against S. pyogenes. In addition, the IZ of 50 and 100 µL of 1 mg·mL⁻¹ AgNPs and 100 µL of 1 mM AgNO₃ solution was found to be 17 ± 1, 16.33 ± 1.15, and 15.67 ± 1.53 mm, respectively against E. coli (ATCC 25922). Besides, no IZ was found for 100 µL of 0.1% aqueous seed extract of T. copticum against E. coli. Moreover, the IZ of 50 and 100 µL of 1 mg·mL⁻¹ AgNPs and 100 µL of 1 mM AgNO₃ solution was found to be 14.67 ± 0.58, 14.33 ± 0.58, and 10.67 ± 1.53 mm, respectively against K. pneumoniae (ATCC 10031). Notably, no IZ was found for 100 µL of 0.1% aqueous seed extract of T. copticum against K. pneumoniae. Notably, no significant difference was found between the IZ of 50 and 100 µL of 1 mg·mL⁻¹ AgNPs in all four tested bacteria (P > 0.05). Besides, the difference between the IZ of 50 and 100 µL of 1 mg·mL⁻¹ AgNPs and 100 µL of 1 mM AgNO₃ solution was significant in S. aureus, S. pyogenes, and K. pneumoniae (P < 0.05). However, no significant difference was found between the IZ of 50 and 100 µL of 1 mg·mL⁻¹ AgNPs and 100 µL of 1 mM AgNO₃ solution in E. coli (P > 0.05).

Figure 6

Well diffusion assay of 100 µL aqueous AgNO₃ solution (0.5 mM), 100 µL aqueous seed extract of T. copticum (0.1%), 50 µL AgNPs (1 mg·mL⁻¹), and 100 µL AgNPs (1 mg·mL⁻¹) against S. aureus, S. pyogenes, E. coli, and K. pneumoniae. Data were presented as mean ± standard deviation (SD). The difference between the groups with no common letter was deemed statistically significant at a 95% confidence level (P < 0.05).

In agreement with our findings, Kelkawi et al. biosynthesized AgNPs using 1 mL of Mentha pulegium aqueous extract. It was revealed that the biological NPs were anisotropic morphologically and their diameter was recorded as 5–50 nm. The antibacterial activity of biogenic NPs was also assessed against S. aureus, S. pyogenes, and E. coli with mean IZ of 9.65 ± 0.64, 10.63 ± 0.67, and 10.20 ± 1.20 mm, respectively. The suggested mechanisms regarding the antibacterial activity of the AgNPs were a surge in permeability and DNA damage as a result of disturbance of the bacterial cell membrane and over generation of ROS. Notably, it was indicated that the existence of a high concentration of biological extract encapsulating the particles could inhibit the proper release of AgNPs. Therefore, AgNPs biosynthesized using low concentrations of M. pulegium were found to be more efficient antibacterial agents [27]. Similarly, Rawat and coworkers reported that the Polygonatum graminifolium-mediated fabricated AgNPs with spherical shape and size between 3 and 15 nm could inhibit the growth of S. aureus and E. coli with IZ of 27 and 16 mm, respectively. Further, the scanning electron microscope (SEM) results exhibited irregular shapes and significant damage to the cell wall in AgNP-treated bacterial cells compared to the control confirming the probable attribution of antibacterial potential to the release of AgNPs through the disrupted bacterial cell membrane [28]. Likewise, Csakvari et al. evaluated the antibacterial potential of the AgNPs, biosynthesized using leaf extract of Cannabis sativa, against gram-positive and negative bacteria. It was reported that sphere-shaped AgNPs with a diameter of <69 nm exhibited the IZ of 10–14 mm for K. pneumonia, 12–13 mm for S. aureus, and 10–12 mm for E. coli. It was hypnotized that due to the thicker layer of peptidoglycan in gram-positive bacteria, the reduction of Ag ions to AgNPs was more effective in gram-negative bacteria. Further, the authors suggested that the mechanisms underlying the antibacterial activity of the NPs were similar disruption of the cell membrane in bacteria, affecting the enzymes in the respiratory chain, and their destructive influence on the bacterial genetic material [34]. The strong inhibitory effect of Abelmoschus esculentus-derived AgNPs against bacterial growth was also confirmed by Devanesan and colleagues using the agar well diffusion method. It was indicated that spherical AgNPs with an average diameter of 16.19 nm and at a dose of 100 µL were able to form IZ of 14 ± 0.5 mm for K. pneumonia, 13.0 ± 1.2 mm for S. aureus and S. pyogenes, and 11.0 ± 1.0 mm for E. coli. It was suggested that Ag ions released after attachment of AgNPs to the bacterial cell wall could lead to the reaction with carboxyl, thiol, and phosphate groups of the amino acids causing the destruction of cell wall and intracellular organelles. Further, production of free radicals, especially ROS by biosynthesized AgNPs, could induce intracellular toxicity and eventually cell death [35]. According to Garibo et al., 5 nm-sized AgNPs biosynthesized utilizing Lysiloma acapulcensis extract could actively combat strains of different pathogenic bacteria. The results of this study revealed that the spherical and quasi-spherical AgNPs were effective against S. aureus, P. aeruginosa, and E. coli with IZ of 16.0 ± 1.0, 15.0 ± 0.5, and 18.0 ± 1.3 mm, respectively. The authors hypothesized that the bacteriostatic effect of the biofabricated AgNPs was attributed to damages induced to the cell membrane and the inability of bacterial DNA to replicate [36]. Moreover, Golabiazar et al. examined the efficacy of the phytofabricated AgNPs against four different strains of bacteria. The mentioned NPs were found to be cubic morphological and their average size was reported as 17–18 nm. The results exhibited that the growth of S. aureus and S. pyogenes as gram-positive bacteria and E. coli and K. pneumonia as gram-negative bacteria were hindered by the AgNPs with IZ ranging from 0.6 to 3 cm. Attachment of the biological AgNPs to the cell membrane of bacteria followed by undermining the membrane function and increased permeability was regarded as the probable role of AgNPs as antibacterial agents [37]. In a similar study, Mariadoss and coworkers reported the herbal-mediated biosynthesis of AgNPs using Malus domestica with a nearly spherical structure and an average size of 50–107.3 nm. These green AgNPs at the concentration of 500 μg·mL⁻¹ revealed IZ of 23 and 8 mm against E. coli and K. pneumonia, respectively [38]. Rautela et al. assessed the antimicrobial activity of the biogenic AgNPs against pathogenic bacteria as well. The seeds extract of Tectona grandis was employed to form AgNPs mostly spherical with a diameter of less than 100 nm confirmed by SEM analyses. It was indicated that strains of S. aureus (17 mm) and E. coli (20 mm) treated with the AgNPs exhibited IZs in the plates. The mechanism by which the NPs hindered the bacterial growth was thought to be the leakage of sugar and proteins from the damaged bacterial membrane resulting in a decrease in the bacterial function [39]. Alternatively, Mahmoodi Esfanddarani and coworkers biofabricated AgNPs by methanol extract of Malva sylvestris and indicated their spherical and hexagonal shape along with a size of between 20 and 40 nm according to TEM results. The acceptable inhibitory effect of the biologically formed AgNPs was recorded against E. coli and S. aureus with IZ of 10.2 ± 0.5 and 10.4 ± 0.5 mm, respectively [40]. In addition, the antibacterial activity of the A. esculentus-derived AgNPs was measured against different bacterial strains. It was reported that the phytofabricated AgNPs were spherical in shape with an average diameter of 20–30 nm and the IZ diameters obtained were 16.27 ± 0.51 mm corresponding to E. coli and 12.47 ± 0.31 mm corresponding to S. aureus. The authors considered oxidative stress caused by over generation of ROS responsible for the antibacterial effect. The disruption of bacterial membrane, DNA, lipids, and proteins was believed to be the main mechanism underlying this process [41]. Moreover, Lateef et al. introduced a similar green route for the production of AgNPs utilizing the extract of cocoa pod husk. The size distribution of the AgNPs was found to be between 4 and 32 nm. It was confirmed that cocoa pod husk-mediated AgNPs possessed a significant antibacterial effect with IZ of 10–14 mm against E. coli and K. pneumonia [42]. Further, Ajah et al. used Haemophilus influenza for the bioreduction of Ag ions and biosynthesized AgNPs with predominantly spherical morphology and an average diameter of 80.05–101.15 nm. The antibacterial effect of the NPs was studied against Streptococcus spp., Klebsiella spp., S. aureus, and E. coli, and the results demonstrated that the environmentally safe AgNPs could form IZ of 15, 24, 31, and 18 mm, correspondingly when the brain heart infusion broth incubated in light used. Although the exact mechanism of inhibition of bacterial growth is yet to be fully addressed, the authors suggested that production of free radicals and ROS, an increase in penetration by binding AgNPs to the external proteins and forming pores, and disruption of DNA replication could play a role in the death of the bacteria [43]. Alternatively, Baghayeri and coworkers reported another approach for the biological manufacturing of AgNPs utilizing an aqueous extract of Salvia leriifolia. The diameter recorded for the NPs was 27 nm on average and they were mostly spherical morphologically. In this study, the antibacterial activity of the NPs was confirmed through well diffusion assay and it was reported that the green AgNPs were able to inhibit the growth of K. pneumonia, Staphylococcus coagulase, S. pneumonia, and E. coli with IZ of 16.33 ± 0.9, 18.67 ± 0.4, 20 ± 0.0, and 14.0 ± 0.7 mm, respectively. The possible antibacterial mechanisms included cellular leakage as a result of more permeability, interference with the proper function of enzymes, proteins, or DNA, and formation of a toxic environment by the generation of free radicals [44]. In a similar experiment, Samuggam et al. reported the biological synthesis of AgNPs with a mean particle size of 17 nm and spherical shape using the extract of Spondias mombin. The AgNPs were found to be suitable antibacterial tools forming IZ with diameters of 20.65 ± 0.35, 23.65 ± 0.35, 22, and 20.65 ± 0.35 mm in the plates consisting of strains of Staphylococcus haemolyticus, Staphylococcus epidermidis, S. aureus, and S. pyogens, respectively. In the case of gram-negative bacteria, the zones of 21 mm corresponding to E. coli and 0.33 mm corresponding to K. pneumonia were observed. The authors hypothesized that ROS formed by the AgNPs were responsible for the conversions in the structure of the bacterial cells leading to inactivation of the bacteria [45]. In a study, Alzubaidi et al. prepared AgNPs using ethanolic extract of flaxseed and reported the structure of the NPs as spherical and their diameter as 46.98 ± 12.45 nm. This study confirmed that the NPs could actively inhibit the growth of four different bacterial species in an environmentally sound manner. The IZ of 11.0 ± 0.42, 10.8 ± 1.02, 8.9 ± 1.08, and 8.7 ± 1.04 mm was observed in the plates for E. coli, K. pneumonia, S. aureus, and S. pyogenes, respectively. Although there was no reliable evidence for any exact mechanism regarding the antibacterial effect of AgNPs, better penetration of NPs into the bacterial cells because of their nano-sized diameter and production of oxidative stress damaging the bacterial function were considered the probable mechanisms [46]. Likewise, Alkhulaifi et al. reported the biosynthesis of AgNPs with spherical shape and an average diameter of 59.74 nm using peels of Citrus limon. These NPs exhibited notable antibacterial activity against E. coli and S. aureus with IZ of both 35 mm [47]. In another study, Citradewi et al. employed an environment-friendly process to prepare the AgNPs and examine their toxic effect on pathogenic bacteria. Cockle shell was utilized to produce AgNPs from Ag ions with irregular spherical morphology and a diameter ranging from 4 to 12 nm. The bactericidal activity of the biogenic NPs was assessed against S. aureus, S. pyogenes, E. coli, and K. pneumonia and it was concluded that the AgNPs were more effective against gram-negative bacteria (i.e., E. coli and K. pneumonia) with wider IZs comparing to gram-positive bacteria naming S. aureus and S. pyogenes [48].

3.2.2 Broth microdilution assay

3.2.2.1 MIC and MBC assessment

As shown in Figure 7a, the MIC values of the AgNPs were found to be 0.25, 1, 0.5, and 0.5 µg·mL⁻¹ against S. aureus, S. pyogenes, E. coli, and K. pneumoniae, respectively. Besides, the MIC values of the standard antibiotic (tetracycline) were found to be 0.25, 0.25, 2, and 0.5 µg·mL⁻¹ against S. aureus, S. pyogenes, E. coli, and K. pneumoniae, respectively. According to the MIC values, E. coli was more sensitive to AgNPs than tetracycline. In contrast, S. pyogenes was more sensitive to tetracycline than AgNPs. The MBC values also confirmed these findings. As shown in Figure 7b, the MBC values of the AgNPs were found to be 8, 16, 4, and 0.5 µg·mL⁻¹ against S. aureus, S. pyogenes, E. coli, and K. pneumoniae, respectively. Besides, the MBC values of the standard antibiotic (tetracycline) were found to be 0.5, 0.5, 8, and 8 µg·mL⁻¹ against S. aureus, S. pyogenes, E. coli, and K. pneumoniae, respectively. As shown in Figure 8, several mechanisms were proposed for the antibacterial activity of AgNPs in the literature. In a study, Barabadi et al. reported the preparation of AgNPs using Zataria multiflora as the biofactory. The NPs were spherical and their hydrodynamic size was recorded as 25.5 nm. The antibacterial activity of biosynthesized AgNPs was measured against S. aureus with MIC of 4 µg·mL⁻¹ compared to commercial AgNPs with MIC of 8 µg·mL⁻¹. The probable mechanisms were found to be the bactericidal influence of Ag ions released after penetration of AgNPs and disturbance of the respiratory chain and bacterial cell wall [26]. Likewise, de Aragão et al. assessed the antibacterial effect of biogenic AgNPs. The polysaccharides of the seaweed Gracilaria birdiae were employed as stabilizing and reducing agents for the formation of spherical AgNPs with a diameter ranging from 20.2 to 94.9 nm. It was exhibited that at the polysaccharide concentration of 0.05% (pH 11), the AgNPs inhibited the growth of S. aureus and E. coli with MIC of 81.2 and 40.6, respectively [49]. Eren and Baran also evaluated the ability of biologically synthesized AgNPs to tackle bacterial growth. It was reported that using leaves of maize (Zea mays L.) could reduce Ag ions to stable AgNPs with a mean size of 12.63 nm and spherical structure. The results regarding the antibacterial potential of the biogenic AgNPs revealed the MIC of 0.337 µg·mL⁻¹ for S. aureus and 0.084 µg·mL⁻¹ for E. coli [50]. Alternatively, Biswas and coworkers conducted research regarding the role of biosynthesized AgNPs as antibacterial agents. It was exhibited that the Solanum viarum-derived AgNPs were mainly spherical and oval morphologically and their particle diameter was between 2 and 40 nm. The green AgNPs were effective against E. coli with MIC of 0.41 ± 0.00 mM, S. aureus susp. aureus with MIC of 0.13 ± 0.01 mM, Streptococcus pneumoniae and K. pneumonia with MIC of more than 1 mM [51]. Similarly, Das Mahapatra et al. investigated the potential of biologically manufactured AgNPs for confronting some pathogenic bacteria. According to this study Oxalis corniculata aqueous extract was able to reduce Ag ions to AgNPs with spherical shape and an average diameter of 40 nm. These ecofriendly NPs at the concentration of 40 µg·mL⁻¹ were tested against S. pyogenes, E. coli, K. pneumonia, and S. aureus with MIC of 0.84, 0.72, 0.78, and 0.76 mM, respectively [52]. Furthermore, in a study AgNPs were biofabricated using Murraya koenigii (L.) with spheroidal morphology and a size ranging from 5 to 20 nm. These biosynthesized AgNPs effectively hindered the growth of extended-spectrum β-lactamase-producing E.coli, control strain of E. coli, methicillin-resistant S. aureus, and methicillin-sensitive S. aureus with MIC of 64, 16, 32, and 32 μg·mL⁻¹, respectively. The possible mechanism by which the green AgNPs tackled the bacterial strains was the release of Ag ions into the bacterial cells followed by disturbance of cell division and respiration function by NPs leading to the destruction of the microorganism [53]. Likewise, Soliman and colleagues used Rhodotorula sp. strain ATL72 as the green source for the synthesis of AgNPs biologically. The results revealed that the size range of the NPs was 8.8–21.4 nm and they were oval and spherical shaped according to TEM results. The efficacy of the biogenic AgNPs was evaluated against strains of E. coli and it was reported that the growth of E. coli was completely inhibited by the AgNPs at the concentration of 1 µg·mL⁻¹. In the case of the mechanism underlying the antibacterial activity, it was thought that the interaction of AgNPs with the thiol groups present in the proteins could cause the unfolding and degradation of the proteins and eventually malfunctioning of different bacterial vital processes, especially translation [54]. Similarly, Altinsoy et al. proposed a green method for the production of AgNPs using E. coli, Saccharomyces cerevisiae, and Bacillus subtilis as reducing agents. The results of characteristic analyses revealed that the size of the NPs ranged from 126 to 323 nm and they possessed various structures. Further, different bacterial strains including S. pneumonia and K. pneumonia were tested and AgNPs were found significantly effective at the concentration of 500 µg·mL⁻¹ by 85% against the mentioned bacteria according to MIC results [55]. Hemmati and colleagues as well examined the antibacterial potential of green-synthesized AgNPs. In this study, they used the aqueous flower extract of Fritillaria for the formation of AgNPs with an average size of 10 nm and mostly spherical morphology. MIC tests exhibited that AgNPs at the concentration of 4, 2, and 1 µg·mL⁻¹ affected the bacterial strains of Staphylococcus saprophyticus, S. pyogenes/S. pneumonia, and K. pneumonia/ E. coli/S. epidermidis/S. aureus, respectively [56]. Accordingly, Mohanta and coworkers biosynthesized AgNPs using three different biofactories including Bridelia retusa, Glochidion lanceolarium, and Semecarpus anacardium. The NPs were characterized as spherical in a size ranging from 52 to 96 nm according to SEM analyses. The toxic effect of biogenic NPs was evaluated against S. aureus and E. coli and in the case of B. retusa MIC values of 64.13 ± 0.3 and 43.94 ± 0.2 µg·mL⁻¹ were recorded, respectively. The AgNPs biosynthesized by S. anacardium inhibited the growth of against S. aureus (33.77 ± 0.2 µg·mL⁻¹) and E. coli (23.49 ± 0.2 µg·mL⁻¹) effectively as well. Further, the MIC values of 43.94 ± 0.2 µg·mL⁻¹ corresponding to S. aureus and 44.02 ± 0.3 µg·mL⁻¹ corresponding to E. coli were recorded for the G. lanceolarium-mediated synthesis of AgNPs. The suggestion regarding the antibacterial activity of the AgNPs was similarly damaging the cell membrane and oxidizing the proteins of plasma membrane. However, it was possible that NPs could block the pores on the membrane leading to a decrease in cell respiration [21]. Similarly, Muthulakshmi and coworkers reported an ecofriendly approach for the preparation of AgNPs and assessed their antibacterial potential against different microorganisms including S. aureus, S. pyogenes, Streptococcus mutans, E. coli, and K. pneumonia. The culture filtrate from Enterobacter cloacae was the biological source of the synthesis to form the AgNPs with a size range of 12–30 nm and spherical structure. MIC values of 6.3, 3.1, 12.5, 3.1, and 6.3 µg·mL⁻¹ were obtained for S. aureus, S. pyogenes, K. pneumonia, S. mutans, and E. coli, respectively. It was suggested that the attachment of Ag ions to DNA and disruption of replication could be responsible for the antibacterial effect of the NPs [57]. Likewise, AgNPs were produced utilizing Wild Ginger (Zingiber zerumbet) extract, and their toxic effect on different bacterial strains was tested. These NPs were spherical according to SEM results and most of the NPs were in a diameter of 153.2 nm. The antibacterial assessments revealed that AgNPs biosynthesized by the aqueous extract of Z. zerumbet at the concentration of 50 µg·mL⁻¹ could tackle S. aureus and S. mutans with MIC values of both 25 µg·mL⁻¹ [58]. Alternatively, Youssef et al. reported the green fabrication of AgNPs using honey as the reducing factor. The biologically formed AgNPs were found to be sphere-shaped and their diameter was between 25 and 70 nm. In this study, the role of the NPs as alternative antibacterial agents was assessed and it was exhibited that E. coli with MIC of 6.3 µg·mL⁻¹ was highly sensitive to the biogenic AgNPs compared to K. pneumonia strains with MIC of 12.5 µg·mL⁻¹. the authors suggested that the formation of reactive Ag ions and the massive surface area could attributed to the antibacterial activity of the NPs [59]. Wypij et al. also focused on the biological synthesis of AgNPs using Streptomyces xinghaiensis OF1 strain as the biofactory. According to TEM analyses the NPs were spherical in shape and 5–20 nm in size. This study confirmed that the green synthesized AgNPs could inhibit the growth of different microorganisms including S. aureus with MIC of 256 µg·mL⁻¹ and E. coli with MIC of 64 µg·mL⁻¹ [60].

Figure 7

(a) MIC and (b) MBC assessment of AgNPs compared to tetracycline as standard antibiotic against S. aureus, S. pyogenes, E. coli, and K. pneumoniae.

Figure 8

Proposed antibacterial mechanisms of colloidal silver particles [The Figure was developed in part using Servier Medical Art, which is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).].

3.2.3 ROS assay

The oxidative-associated antibacterial mechanism of AgNPs was measured by evaluating the ROS level of bacterial supernatant after one-night incubation with AgNPs at the concentration of MIC and 20*MIC in comparison to standard antibiotic (tetracycline) with the same concentrations. The supernatant of nontreated bacteria with the same conditions was considered as negative control. As shown in Figure 9, the results showed a significant dose-dependent increase in the ROS concentration of AgNP-treated samples with the concentrations of MIC and 20*MIC against S. aureus, E. coli, and K. pneumoniae (P < 0.05). However, no significant difference was found between the ROS concentration of AgNP-treated samples with the concentrations of MIC and 20*MIC against S. pyogenes. In addition, a significant dose-dependent increase was found in the ROS concentration of tetracycline-treated samples with the concentrations of MIC and 20*MIC against S. aureus, S. pyogenes, and K. pneumoniae (P < 0.05). However, no significant difference was found between the ROS concentration of tetracycline-treated samples with the concentrations of MIC and 20*MIC against E. coli. Overall, both AgNPs and tetracycline enhanced the generation of ROS significantly in all studied bacteria compared to the control (P < 0.05) suggesting an oxidative-associated antibacterial mechanism. According to the literature, the intracellular oxidative stress causes damages to the bacterial cell wall and intracellular moieties containing DNA, proteins, and lipids leading to cell death [61]. In a similar study, the colloidal silver particles were phytosynthesized by employing leaf extract from Ocimum gratissimum with a hydrodynamic particle size of 31 nm and a zeta potential of −15 mV. The O. gratissimum-derived AgNPs showed significant antibacterial activity against resistant strains of E. coli and S. aureus and enhanced their intracellular ROS generation by 85.68% and 89.36%, respectively. The authors reported that the AgNP-induced oxidative damage is one of the main antibacterial mechanisms of AgNPs [62]. Likewise, the phytofabricated spherical-shaped AgNPs by using the aqueous extract of aerial parts of Astragalus spinosus in the range of 30–40 nm showed significant antibacterial activity against Actinomyces viscosus and Streptococcus mutans. The ROS assay revealed that the green-synthesized AgNPs increased the ROS levels in both tested bacteria compared to the control group [63]. Alternatively, plant-mediated synthesis of uniformly spherical-shaped AgNPs was reported by using the leaf extract of Allophylus cobbe with an average hydrodynamic particle size of 5  ±  4 nm. The phytosynthesized AgNPs showed a great bactericidal performance and increased the intracellular overgeneration of ROS alone and in combination with standard antibiotics in both tested gram-negative and gram-positive bacteria [64].

Figure 9

ROS assessment of AgNPs compared to tetracycline as a standard antibiotic at the concentrations of MIC and 20*MIC against S. aureus, S. pyogenes, E. coli, and K. pneumoniae. Data was presented as mean ± standard deviation (SD). The difference between the groups with no common letter was deemed statistically significant at a 95% confidence level (P < 0.05).

3.2.4 Flow cytometry-based quantitative assessment of cell viability

Flow cytometric analysis represented a dose-dependent bacterial cell death in the AgNP-treated samples against S. aureus (Figure 10a), S. pyogenes (Figure 10b), E. coli (Figure 10c), and K. pneumoniae (Figure 10d). As shown in Figure 10a, bacterial cell death of 0.985% (a), 20.3% (b), 47.1% (c), and 55.4% (d) was found in the control group (nontreated bacterial sample) and in the AgNP-treated cells at the concentrations of MIC (0.25 µg·mL⁻¹), 10*MIC (2.5 µg·mL⁻¹), and 20*MIC (5 µg·mL⁻¹), respectively, against S. aureus. Besides, as shown in Figure 10b, bacterial cell death of 1.07% (a), 17.4% (b), 39.3% (c), and 52.8% (d) was found in the control group (nontreated bacterial sample) and the AgNP-treated cells at the concentrations of MIC (1 µg·mL⁻¹), 10*MIC (10 µg·mL⁻¹), and 20*MIC (20 µg·mL⁻¹), respectively, against S. pyogenes. Moreover, as shown in Figure 10c, bacterial cell death of 0.813% (a), 16.1% (b), 47.1% (c), and 64.6% (d) was found in the control group (nontreated bacterial sample) and the AgNP-treated cells at the concentrations of MIC (0.5 µg·mL⁻¹), 10*MIC (5 µg·mL⁻¹), and 20*MIC (10 µg·mL⁻¹), respectively, against E. coli. Furthermore, as shown in Figure 10d, bacterial cell death of 0.453% (a), 17% (b), 38.5% (c), and 61.9% (d) was found in the control group (nontreated bacterial sample), and the AgNP-treated cells at the concentrations of MIC (0.5 µg·mL⁻¹), 10*MIC (5 µg·mL⁻¹), and 20*MIC (10 µg·mL⁻¹), respectively, against K. pneumoniae. In a similar study, the plant-mediated synthesized AgNPs from Aloe arborescens with an average size of 38 ± 2 nm showed significant antibacterial activity against Pseudomonas aeruginosa and S. aureus with MIC values of 8 and 12 µg·mL⁻¹, respectively. Then, the flow cytometry analysis was performed to compare the percentage of dead cells in AgNP-treated samples at the concentration of MIC. The findings showed 66.8% and 25.5% cell death in AgNP-treated P. aeruginosa and S. aureus at the concentration of 8 and 12 µg·mL⁻¹ over 12 h incubation at 37°C, respectively [65]. Alternatively, flow cytometry analysis was conducted to evaluate the cell viability of the phytofabricated AgNP-treated E. coli and S. aureus. The findings showed 89.24% and 93.62% cell death against AgNP-treated E. coli and S. aureus at the concentration of 4 and 8 µg·mL⁻¹ over 12 h incubation at 37°C, respectively [62]. Similarly, flow cytometry analysis was conducted to evaluate the cell viability of the phytofabricated AgNP-treated P. aeruginosa. The flow cytometric susceptibility assay showed 94.68% cell death against AgNP-treated P. aeruginosa at the concentration of 32 µg·mL⁻¹ over 12 h incubation at 37°C [66].

Figure 10

Flow cytometry-based quantitative assessment to compare cell viability in the bacterial samples that were treated with AgNPs against ((a) S. aureus, (b) S. pyogenes, (c) E. coli, and (d) K. pneumoniae. (a) Nontreated sample (control), (b) AgNP-treated sample at MIC concentration, (c) AgNP-treated sample at 10*MIC concentration, and (d) AgNP-treated sample at 20*MIC concentration).

Significantly, flow cytometric analysis represented a dose-dependent bacterial cell death in the tetracycline-treated samples against S. aureus (Figure 11a), S. pyogenes (Figure 11b), E. coli (Figure 11c), and K. pneumoniae (Figure 11d). As shown in Figure 11a, bacterial cell death of 0.985% (a), 18% (b), 41.2% (c), and 51.6% (d) was found in the control group (nontreated bacterial sample) and the tetracycline-treated cells at the concentrations of MIC (0.25 µg·mL⁻¹), 10*MIC (2.5 µg·mL⁻¹), and 20*MIC (5 µg·mL⁻¹), respectively, against S. aureus. Besides, as shown in Figure 11b, bacterial cell death of 1.07% (a), 11.9% (b), 29% (c), and 46.5% (d) was found in the control group (nontreated bacterial sample) and the tetracycline-treated cells at the concentrations of MIC (0.25 µg·mL⁻¹), 10*MIC (2.5 µg·mL⁻¹), and 20*MIC (5 µg·mL⁻¹), respectively against S. pyogenes. Moreover, as shown in Figure 11c, bacterial cell death of 0.813% (a), 12.7% (b), 38% (c), and 58.8% (d) was found in the control group (nontreated bacterial sample) and the tetracycline-treated cells at the concentrations of MIC (2 µg·mL⁻¹), 10*MIC (20 µg·mL⁻¹), and 20*MIC (40 µg·mL⁻¹), respectively, against E. coli. Furthermore, as shown in Figure 11d, bacterial cell death of 0.453% (a), 13.1% (b), 30.6% (c), and 57.6% (d) was found in the control group (nontreated bacterial sample) and the tetracycline-treated cells at the concentrations of MIC (0.5 µg·mL⁻¹), 10*MIC (5 µg·mL⁻¹), and 20*MIC (10 µg·mL⁻¹), respectively, against K. pneumoniae.

Figure 11

Flow cytometry-based quantitative assessment to compare cell viability in the bacterial samples that were treated with tetracycline against ((a) S. aureus, (b) S. pyogenes, (c) E. coli, and (d) K. pneumoniae. (a) Nontreated sample (control), (b) tetracycline-treated sample at MIC concentration, (c) tetracycline-treated sample at 10*MIC concentration, and (d) tetracycline-treated sample at 20*MIC concentration).