*2.5. In Vitro Antibacterial Activity*

#### 2.5.1. In Vitro Susceptibility Testing

The green-synthesized AgNPs exhibited antibacterial activity against *S. aureus* clinical isolates, as they resulted in clear zones around the AgNPs discs by disc diffusion method. The broth microdilution method was utilized to identify the MIC values of AgNPs, and they ranged from 4 to 64 µg/mL. All the following tests were carried out after treatment of the tested isolates with 0.5 MIC values.

#### 2.5.2. Time Kill Curve

The number of colony-forming units (CFU) per milliliter was reduced by more than 3 log units after incubation of *S. aureus* cells for 2 h with 4× MIC and 1 h with 8× MIC in 58.34% and 47.92% of the isolates, respectively. A representative example for the reduction of the CFU/mL is shown in Figure 8.

**Figure 8.** Time kill plot of (**a**) 4× MIC and (**b**) 8× MIC of the green synthesized AgNPs against *S. aureus* isolates.

#### 2.5.3. Membrane Integrity and Permeability

We investigated the cell membrane integrity of *S. aureus* isolates after treatment with the green synthesized AgNPs (at concentrations equal to 0.5 MIC values) via detection of the release of the materials (DNA and RNA), which absorb at 260 nm, from the bacterial isolates. Herein, we found that the membrane integrity significantly decreased (*p* < 0.05) in 45.8% of the isolates after treatment with AgNPs. Figure 9a illustrates a representative example.

**Figure 9.** Line chart showing (**a**) the cell membrane integrity and (**b**) the membrane permeability of a representative *S. aureus* isolate before and after treatment with the green-synthesized AgNPs (at concentrations equal to 0.5 MIC values).

When the bacterial membrane permeability increases, *O*-nitrophenyl-β-galactopyranoside (ONPG) enters the bacterial cytoplasm in a large amount. In the cytoplasm, ONPG is broken down into *O*-nitrophenol (ONP) by a β-galactosidase enzyme that is present in the cytoplasm. Thus, the membrane permeability was tracked by monitoring the absorbance at OD<sup>420</sup> (the yellow color of ONP can absorb at 420 nm) with time. The membrane permeability significantly increased (*p* < 0.05) in 56.25% of *S. aureus* isolates after treatment with AgNPs and an illustrative example is revealed in Figure 9b.

#### 2.5.4. Membrane Depolarization

Membrane depolarization was determined in the tested isolates using DiBAC4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol) fluorescent stain. This is a membrane potential-sensitive stain that can enter the depolarized cell cytoplasm and bind to the intracellular proteins exhibiting an enhanced fluorescence. In the current investigation, we noticed that treatment with AgNPs exhibited a considerable reduction (*p* < 0.05) in the membrane potential in 35.42% of *S. aureus* isolates. A demonstrative example of the decrease in the membrane potential after AgNPs treatment is presented in Figure 10.

#### 2.5.5. SEM Examination

The ultrastructural and morphological changes of *S. aureus* cells treated with the greensynthesized AgNPs were observed by SEM (Figure 11). The electron micrographs obtained by SEM revealed that the untreated cells had sphere-shaped, intact, smooth surfaces. On the other hand, the treated cells had a deformed and distorted shape.

#### 2.5.6. Efflux Activity

The efflux activity of the tested *S. aureus* isolates was assessed by testing the capability of the cells to pump out ethidium bromide (EtBr) to the surrounding medium by the EtBr cartwheel method. Herein, we categorized the efflux activity of *S. aureus* isolates into three classes; negative, intermediate, and positive efflux activity as presented in Table 2. Eleven (22.92%) *S. aureus* isolates exhibited a reduction in their efflux activity when treated with the green-synthesized AgNPs. The efflux activity of these isolates changed from positive to either intermediate or negative.

#### 2.5.7. Quantitative Real-Time PCR (qRT-PCR)

qRT-PCR was utilized for more in-depth exploration of the impact of the greensynthesized AgNPs on the efflux pump activity of the tested isolates. *S. aureus* bacteria

(*n* = 11) that displayed a decline in their efflux pump activity by the EtBr cartwheel method were selected for this assay. We found that the transcriptional levels of *nor*A, *nor*B, and *nor*C efflux pump genes decreased in 72.73%, 45.45%, and 18.18% of the tested isolates, respectively, after treatment with AgNPs. The fold changes mean values of *nor*A, *nor*B, and *nor*C efflux pump genes ranged from 0.11 to 0.47, respectively, as presented in Figure 12.

**Figure 10.** Flowcytometric chart (**a**) dot plot, (**b**) histogram (fluorescent gap = 67.1%) before treatment, (**c**) dot plot, and (**d**) histogram (fluorescent gap = 35.1%) after treatment of a representative *S. aureus* isolate with the green-synthesized AgNPs.

**Figure 11.** Scanning electron micrograph of a representative *S. aureus* isolate (**a**) before treatment and (**b**) after treatment with the green-synthesized AgNPs.

#### *2.6. In Vivo Antibacterial Activity*

The impact of AgNPs was investigated on macroscopic healing and skin histology following excisional wound healing as follows.


**Table 2.** Efflux activity of *S. aureus* isolates determined using EtBr cartwheel method, before and after treatment with AgNPs.

\* Concentration of EtBr at which *S. aureus* bacteria started to produce fluorescence. The isolates which emit fluorescence at 0.5 mg/L, 1–2 mg/L, and 2.5 mg/L lack efflux activity, have intermediate efflux activity, and have positive efflux activity, respectively.

**Figure 12.** Bar charts showing the transcriptional level fold changes of (**a**) *nor*A, (**b**) *nor*B, and (**c**) *nor*C genes after treatment with the green-synthesized AgNPs. \* represents a significant decrease in the fold change.

### 2.6.1. Macroscopic Healing

The rates of the macroscopic wound healing of the studied groups were inspected on days 0, 3, and 7, considering the day of the wound creation as day 0. Betadine™ and AgNPs groups demonstrated full and notable wound healing in comparison with the control group (Figure 13).

**Figure 13.** Macroscopic examination of the wound healing of the control, Betadine™, and AgNPs rat groups over the days 0, 3, and 7 starting from the day of wound creation (day 0).

Betadine™ and AgNPs groups exhibited significant wound healing on day 3 with wound healing percentages of 90.19 and 92.3, respectively, compared to the control group. Furthermore, they exhibited full wound healing on day 7 (with wound healing percentages of 99.02 and 99.23, respectively, in comparison with the control group (Figure 14a).

In addition, both Betadine™ and AgNPs groups exhibited a significant decrease in CFU/mL in comparison with the control group (Figure 14b).

#### 2.6.2. Histological Examination

The skin section of the wounds of the control group showed a wide area of epidermal loss and infiltration with inflammatory cells with granulation tissue formation (Figure 15a,d).

**Figure 15.** Histological examination of the wounds of rats on the seventh day. The control group (**a**,**d**) showed the presence of a wide area of epidermal loss in addition to ulceration in the wound gap (black arrow) with adjacent thickening in the epidermis (arrowhead). There was a highly cellular granulation tissue that is rich in inflammatory cells (black star). The Betadine™ group (**b**,**e**) showed an intact thin epidermal layer with intact keratinocytes and subcellular details (arrow). There was an intact layer of the dermis with a normal distribution of cellular elements and abundant well-organized collagen fibers (black star). The AgNPs group (**c**,**f**) showed efficient wound healing with complete epidermal re-epithelialization as well as wound closure (black arrow). There was more abundant fibroblastic activity with dermal granulation tissue rich in collagen (dashed arrow).

On the other hand, the skin section of the wounds of the Betadine™ group showed enhanced epidermal re-epithelialization and wound closure with more abundant fibroblastic activity and more collagen-rich dermal granulation tissue (Figure 15b,e). The section of the AgNPs treated group also exhibited complete wound healing with continuous epidermis and underlying fibrosis and collagenosis (Figure 15c,f).

#### **3. Discussion**

The widespread community and hospital-acquired infections caused by *S. aureus* isolates are a global consideration. In addition, these pathogenic bacteria are largely related to resistance to many commercially available antimicrobial agents [17]. Thus, many researchers have focused their studies on the exploration of new antimicrobial compounds against various types of pathogenic bacteria such as *S. aureus*. Natural products such as plants are showing promising antimicrobial activity with relatively low toxicity, low cost, and high bioavailability [18–23]. Herein, we used GTLE for the green synthesis of AgNPs. AgNPs drew our attention owing to their documented versatile activities in the literature, such as their antimicrobial and anti-inflammatory properties in addition to their wound healing promotion capability. AgNPs are currently utilized as interesting tools to face many emerging therapeutic challenges [24]. Despite their advantageous properties, the synthesis of AgNPs can be a high-cost process and a harmful approach. This is because of the utilization of chemical compounds and the possible production of certain harmful by-products [25–27]. Therefore, we decided to rely on the green synthesis of AgNPs to avoid these drawbacks. Many natural products such as plant extracts can be used in the green biosynthesis of AgNPs. In this case, the bioactive compounds of plants reduce silver to form AgNPs. In this way, we avoid the use of chemical reducers with their

accompanying problems. The fundamental principle of the green methods is to utilize nontoxic biomolecules for the synthesis of nanoparticles via the reduction of metal ions in an aqueous solution. Most biomolecules such as DNA, proteins, and enzymes are quite expensive, easily decomposed, and vulnerable to being contaminated. On the other hand, many plant extracts are available, affordable, and stable against most environmental conditions (such as pH, temperature, and salt concentration) [5,28,29].

Nontoxic, environmentally friendly methods were used to synthesize bioinspired silver nanoparticles from GTLE. The quantification of the flavonoids and phenolic acids of *G. thailandica* was evaluated by the HPLC-DAD technique using 20 standard compounds. A few studies have documented the presence of these types of bioactive compounds in different *Gardenia* species. The HPLC-DAD analysis detected major phenolic compounds that are reported to possess antitumor, antibacterial, antioxidant, antidiabetic, and antihypercholesterolemic activities through different pathways [13]. Six phenolic acids were recognized (chlorogenic, rosmarinic, cinnamic, vanillic, *p*-coumaric, and syringic acid). In addition, four flavonoids were identified (quercetin-3-rutinoside, apigenin-7-glucoside, luteolin, and chrysin). The results of the HPLC-DAD analysis of *G. thailandica* revealed the presence of quercetin-3-rutinoside at a concentration of 2477.37 µg/g as the major flavonoid glycoside, while chlorogenic acid was the major phenolic acid in the extract at a concentration of 1441.03 µg/g, followed by rosmarinic acid at a concentration of 796.67 µg/g.

The antibacterial activity of the green-synthesized AgNPs was investigated both in vitro and in vivo. AgNPs exhibited antibacterial activity against *S. aureus* clinical isolates with MIC values that ranged from 4 to 64 µg/mL. Many studies have recorded that greensynthesized AgNPs have antibacterial activity against different pathogenic bacteria [30–33]. The short reproductive time of *S. aureus* bacteria is one of the principal reasons for the infectivity of such pathogenic bacteria [30]. Therefore, we investigated the impact of AgNPs on the time-kill curve of the tested *S. aureus* isolates and we found that the CFU/mL of *S. aureus* isolates was reduced by more than 3 log units after its incubation for 2 h with 4 × MIC and 1 h with 8 × MIC in 58.34% and 47.92% of the isolates, respectively.

As the bacterial cell membrane is an important target for several antimicrobials, we investigated the impact of the green-synthesized AgNPs on membrane characteristics including the membrane integrity, permeability, and depolarization. The bacterial cell membrane is considered to be a barrier with a selective permeability character, and the loss of this property can lead to cell death [34]. Herein, we investigated the membrane integrity of the tested bacteria before and after treatment with AgNPs by observing the leakage of materials absorbing 260 nm over time. We observed that treatment with AgNPs resulted in a massive reduction (*p* < 0.05) in the membrane integrity in 45.8% of the isolates. Many different techniques can be used for the evaluation of membrane permeability. In the current study, we used the ONPG method, which relies on the concept that when the bacteria are losing the ability to control their membrane permeability, the penetration of this compound increases [34]. Our results showed that the membrane permeability of the tested bacterial cells significantly increased (*p* < 0.05) in 56.25% of the isolates after treatment with AgNPs. Owing to the importance of the membrane potential in bacterial viability, we used DiBAC4, a fluorescent probe that enters the cell and links to the intracellular proteins when the membrane potential is lost. Here, the green-synthesized AgNPs resulted in a considerable reduction (*p* < 0.05) in membrane potential in 35.42% of *S. aureus* isolates.

SEM is widely utilized in microbiological research to study the different changes that occur in the ultrastructure and morphology of the bacterial cells when they are treated with antimicrobial agents [35]. Consequently, we used SEM in this study to explore the cell surface characters and external cell morphology to gain the benefit of the higher resolution of SEM when compared to light microscopes. Herein, we noticed that the AgNP-treated bacterial cells had a deformed and distorted shape in comparison with the non-treated ones.

The function of efflux pump proteins is to transfer harmful substances out of bacterial cells [26]. Therefore, efflux pumps are an important resistance mechanism to many antibiotics. In the current study, 22.92% of *S. aureus* isolates presented a reduction in their efflux

activity after treatment with AgNPs. Efflux pumps in *S. aureus* bacteria are encoded by *nor*A, *nor*B, and *nor*C genes. For further elucidation of the effect of AgNPs on the efflux activity of the 11 *S. aureus* isolates that displayed a decline in their efflux activity by EtBr cartwheel assay, qRT-PCR was utilized. We noticed that treatment with AgNPs resulted in a substantial decrease in the expression of *nor*A, *nor*B, and *nor*C genes in 72.73%, 45.45%, and 18.18% of the tested isolates, respectively. Generally, metal nanoparticles could inhibit the efflux pump activity of bacteria by two mechanisms. The first possible mechanism is by direct binding to the efflux pumps' active site and the second mechanism is by disturbing the efflux kinetics [36].

The process of wound healing is associated with certain biological events such as re-epithelialization, fibroplasia in addition to extracellular matrix production. Many natural agents were found to produce satisfactory results in wound healing when compared to the chemical compounds, with the advantages of low cost and low toxicity [37]. Consequently, we used GTLE to synthesize AgNPs and investigated their effect on wound healing in rats with wounds infected with *S. aureus* isolates after seven days of treatment. The group treated with AgNPs exhibited notable wound healing when compared to the other groups. On the histological level, the AgNP-treated group displayed accelerated wound healing with complete epidermal re-epithelialization, abundant fibroblastic activity, formation of collagen-rich dermal granulation tissue, and minimal infiltration of inflammatory cells.

### **4. Materials and Methods**

#### *4.1. Plant Materials and Extract Preparation*

*Gardenia thailandica* Tirveng. leaves were collected from a private garden on the Egypt Alexandria desert road. Esraa Ammar (Plant Ecology, Botany Department, Faculty of Science, Tanta University) confirmed the plant's identification. A voucher sample (PGA-GT-128-W) was maintained in the Tanta University Department of Pharmacognosy's herbarium. The powdered plant (650 g) was extracted with methanol using a maceration method (3 × 5 L). The extract was concentrated using a rotary evaporator to obtain a residue (7.89 g).

#### *4.2. Drugs and Chemicals*

All the chemicals and solvents used in this study were bought from Sigma-Aldrich (St. Louis, MO, USA) and were of high analytical quality.

#### *4.3. HPLC-DAD of GTLE*

An autosampler and a diode-array detector are included in the Agilent Technologies 1100 series liquid chromatography.

The analytical column was an Eclipse XDB-C18 (150 × 4.6 µm; 5 µm) with a C18 guard column (Phenomenex, Torrance, CA, USA). Acetonitrile (solvent A) and 2% acetic acid in water (Solvent B) made up the mobile phase. The flow rate was held constant at 0.8 mL/min for a total run of 70 min, and the gradient program was as follows: 100% B to 85% B in 30 min, 85% B to 50% B in 20 min, 50% B to 0% B in 5 min, and 0% B to 100% B in 5 min. The injection volume was 50 µL, and peaks for benzoic acid, cinnamic acid derivatives, and flavonoids were found simultaneously at 280, 320, and 360 nm, respectively. All samples were filtered using a 0.45 µm Acrodisc syringe filter (Gelman Laboratory, Michigan, USA) before injection. The peaks were identified using congruent retention durations and UV spectra, which were then compared to the standards.

#### *4.4. Green Synthesis of AgNPs*

One millimolar of an aqueous solution of silver nitrate (AgNO3) was prepared and maintained in a cool dark area. For reduction of Ag+ ions, 10 mL of GTLE was added separately into 90 mL of an aqueous solution of 1 mM AgNO<sup>3</sup> and incubated overnight at room temperature in a dark area. The development of AgNPs was indicated by the production of a yellowish-brown color. The produced solutions were directly subjected

to TEM and UV measurements. Centrifugation at 4000 rpm for 30 min was followed by a series of washing in distilled water and filtration to obtain pure AgNPs. The pure AgNPs were further characterized by FTIR, HR-TEM, XRD, zeta potential, and SEM [38–41].

#### *4.5. Characterization of AgNPs*

#### 4.5.1. UV-Vis Spectroscopy

UV-Vis spectroscopy of the green-synthesized AgNPs was monitored using a UV–Vis spectrophotometer (Shimadzu, Kyoto, Japan) after dilution with distilled water.

#### 4.5.2. FTIR

The different functional groups of the produced AgNPs were measured by FTIR spectrometer (Jasco, Tokyo, Japan) in the range of 4000–400 cm−<sup>1</sup> .

#### 4.5.3. HR-TEM

The morphology of the particles (shape and dimensions) in addition to SAED were examined by TEM. (JEOL-JEM-1011, Kyoto, Japan) and HR-TEM at 200 kV (JEOL-JEM-2100, Kyoto, Japan). Three milliliters of the sample were placed on the copper grid for TEM and HR-TEM examination and allowed to dry at room temperature for 15 min.

#### 4.5.4. Zeta Potential and DLS

Particle size, homogeneity of distribution, and zeta potentials of AgNPs were examined using a zeta sizer nano ZN (Malvern Panalytical Ltd., England, UK). Before the measurements, an aliquot of nanoparticles was diluted with ultra-purified water and then sonicated for 15 min.

#### 4.5.5. XRD

The XRD analysis was performed as a surface chemical analysis tool for the characterization of metal nanoparticles [42]. An XPERT-PRO-PANalytical Powder Diffractometer (PAN-alytical B.V., Almelo, The Netherlands) was used to perform XRD utilizing a monochromatic radiation source Cu-K α radiation (θ = 1.5406 Å) at 45 kV and 30 mA at ambient temperature. The silver nano-powder intensity data were gathered over a 2θ range of 4.01◦–79.99◦ .

#### 4.5.6. SEM

The morphology of the biosynthesized AgNPs was observed using SEM (TM1000, Hitachi, Chiyoda, Japan) as described previously [43].

#### *4.6. Determination of the Total Content of Flavonoids and Polyphenols*

The total flavonoid concentration was determined by colorimetric analysis of serial dilutions of the extract using the aluminum chloride technique and quercetin as a reference [44]. Using the Folin–Ciocalteu technique and gallic acid as a reference, the total content of polyphenols was determined [45]. The measured contents were expressed as mg/g equivalent of the corresponding standard for each method.

#### *4.7. Antioxidant Activity of GTLE*

#### 4.7.1. The DPPH Radical Scavenging Capacity

The DPPH radical scavenging capacity of GTLE was evaluated according to the method of Boly et al. [46]. The decrease in DPPH color intensity was measured at 540 nm using the following equation:

> *Percentage inhibition* = = (Average absorbance of blank−average absorbance of the test) Average absorbance of blank × 100

The value of IC<sup>50</sup> was calculated as previously described [47].

#### 4.7.2. The ABTS Radical Scavenging Capacity

The assay was performed as previously reported [48]. Using the linear regression equation taken from the calibration curve, the results are presented as µM Trolox equivalents (TE)/ mg samples (linear dose-inhibition curve of Trolox).

#### 4.7.3. FRAP Assay

The ferric reducing ability assay was conducted according to the method of Benzi et al. [49]. The result is expressed as µM TE/mg sample using the linear regression equation derived from the calibration curve (linear dose-response curve of Trolox).

#### *4.8. In Vitro Antibacterial Activity*

#### 4.8.1. Bacterial Isolates

A total of 48 *S. aureus* clinical isolates were acquired from Tanta University Hospital. *S. aureus* isolates were microscopically examined and were biochemically identified as previously described [50]. *Staphylococcus aureus* (ATCC 29231) was utilized as a reference strain.

#### 4.8.2. Susceptibility Testing

#### Disk Diffusion Method

The antimicrobial activity of AgNPs against *S. aureus* clinical isolates was performed using the Kirby–Bauer disk diffusion method [51]. Mueller–Hinton agar (MHA) (Merck, Germany) plates were inoculated with the bacterial isolates using sterile swabs. Sterile discs were thoroughly saturated with vancomycin and sterile water as positive and negative controls, respectively. In addition, a third disc was saturated with the green-synthesized AgNPs was added. Then, the disks were located on the MHA plates and incubated for 24 h at 37 ◦C. The formed inhibition zones were observed indicating antibacterial activity.

#### Minimum Inhibitory Concentration (MIC) Determination

The MIC values of the green-synthesized AgNPs were determined in a 96-well microdilution plate using the broth microdilution method [52]. The green-synthesized AgNP solution (500 µg/mL) was twofold diluted with each bacterial inoculum in 100 µL of MHB (10<sup>6</sup> CFU/mL). Each microtitration plate had a negative control (MHB only) and positive control (MHB containing bacteria). Each well of the microtitration plate was loaded with 30 µL of the resazurin solution and then the plates were incubated at 37 ◦C for 24 h. The variations in the color were detected.

#### 4.8.3. Time Kill Curve

This was performed as previously reported [53]. Briefly, the green-synthesized AgNPs solution was diluted by MHB containing the bacterial suspensions to obtain a final concentration of 0× MIC, 0.5× MIC, 1× MIC, 2× MIC, 4× MIC, and 8× MIC for each bacterial isolate. The obtained cultures were then incubated in a shaking incubator at 37 ◦C. Aliquots of the cultures (100 µL) were distributed on the surface of MHA plates at 0, 0.25, 0.5, 1, 2, and 4 h. After incubation at 37 ◦C for 24 h, the colonies detected on the MHA plates were quantified in CFU/mL.

#### 4.8.4. Membrane Integrity and Permeability

#### Membrane Integrity Assay

The effect of the green-synthesized AgNPs on the integrity of the cell membrane of the tested isolates was studied by monitoring the release of materials that have absorbance at 260 nm (A260) [54]. In brief, the optical density (OD) of the overnight bacterial cultures in nutrient broth was adjusted to be 0.4 at 630 nm. Then, the bacterial suspensions were centrifuged at 11,000× *g* for 10 min and the obtained pellets were resuspended in 0.5% NaCl solution and their absorbance was adjusted to 0.7 at 420 nm. The membrane integrity was assessed by checking the discharge of materials that have absorbance at 260 nm from the bacterial cytoplasm to the surrounding media over time using a UV/Vis spectrophotometer (SHIMADZU, Kyoto, Japan).

#### Membrane Permeability Assay

Membrane permeability was explored by quantifying the exit of a β-galactosidase enzyme from the bacterial cytoplasm using the substrate of the enzyme (ONPG) [55]. Briefly, 2% lactose was added to the overnight bacterial suspension in nutrient broth. This mixture was then centrifuged, and the obtained pellet was thoroughly rinsed using phosphatebuffered saline (PBS) and resuspended in NaCl solution (0.5%). Finally, each bacterial suspension (1.6 mL) was supplemented with 150 µL of ONPG solution (34 mM). The produced ONP was detected over time using an ELISA reader (Sunrise Tecan, Männedorf, Switzerland) to monitor the increase in absorbance at 420.

#### 4.8.5. Membrane Depolarization

This test was carried out using DiBAC4(3), a fluorescent stain used for staining the tested bacterial cells (both treated and untreated with the green-synthesized AgNPs) [56]. A FACSVerse flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) was used to analyze the staining of the cells.

#### 4.8.6. SEM

The morphological changes of the AgNPs treated *S. aureus* isolates in comparison with the non-treated ones were inspected by SEM (Hitachi, Chiyoda, Japan) as described by McDowell and Trump [57].

#### 4.8.7. Efflux Activity

Efflux activity was tested by the EtBr cartwheel method [58] before and after treatment with AgNPs (at 0.5 MIC values) using the reference strain as a negative control. In brief, bacterial suspensions were inoculated as redial lines onto tryptic soy agar (TSA) plates using swabs and were incubated at 37 ◦C for 18 h. The TSA plates were supplied with EtBr (with concentrations that ranged from 0.5 to 2.5 mg/L). After incubation, the lowest EtBr concentrations that led to fluorescence production by the bacterial isolates were recorded by UV-Vis spectrophotometer (SHIMADZU, Kyoto, Japan). *S. aureus* isolates were then classified according to the recorded EtBr minimum concentrations as follows: isolates with no efflux activity are those that emitted fluorescence at an EtBr concentration of 0.5 mg/L, isolates with intermediate efflux activity are that emitted fluorescence at an EtBr concentration of 1–2.0 mg/L, and isolates with positive efflux activity are those that emitted fluorescence at an EtBr concentration of 2.5 mg/L.

#### 4.8.8. qRT-PCR

We used qRT-PCR for detection of the expression levels of the genes encoding efflux pumps (*nor*A, *nor*B, and *nor*C) in *S. aureus* isolates after treatment with AgNPs. In brief, total RNA was extracted from the pellets of overnight cultures of *S. aureus* isolates using the Purelink™ RNA Mini Kit (Thermo Scientific, Waltham, USA) according to the instructions of the manufacturer. The extracted RNA was then converted into cDNA by power™ cDNA synthesis kit (iNtRON Biotechnology, Seoul, Korea) as described by the manufacturer. Rotor-Gene Q 5plex (Qiagen, Hilden, Germany) was used for performing qRT-PCR for the calculation of the efflux pump gene expression fold changes. The sequences of the utilized primers in addition to the sequence of the housekeeping gene (16S rRNA) primer are presented in Table S1 [59,60]. The levels of the relative expression of the tested genes were quantified by the 2−∆∆Ct method, considering the gene expression levels in the isolates before treatment to be 1 [61]. The statistically significant fold changes were those with two or more-fold changes (either increasing or decreasing) [62].
