*Article* **In Vivo and In Vitro Antimicrobial Activity of Biogenic Silver Nanoparticles against** *Staphylococcus aureus* **Clinical Isolates**

**Nashwah G. M. Attallah 1,†, Engy Elekhnawy 2,\* ,† , Walaa A. Negm 3,\* , Ismail A. Hussein <sup>4</sup> , Fatma Alzahraa Mokhtar <sup>5</sup> and Omnia Momtaz Al-Fakhrany <sup>2</sup>**


**Abstract:** *Staphylococcus aureus* can cause a wide range of severe infections owing to its multiple virulence factors in addition to its resistance to multiple antimicrobials; therefore, novel antimicrobials are needed. Herein, we used *Gardenia thailandica* leaf extract (GTLE), for the first time for the biogenic synthesis of silver nanoparticles (AgNPs). The active constituents of GTLE were identified by HPLC, including chlorogenic acid (1441.03 µg/g) from phenolic acids, and quercetin-3-rutinoside (2477.37 µg/g) and apigenin-7-glucoside (605.60 µg/g) from flavonoids. In addition, the antioxidant activity of GTLE was evaluated. The synthesized AgNPs were characterized using ultravioletvisible spectroscopy, Fourier-transform infrared spectroscopy, transmission and scanning electron microscopy (SEM), zeta potential, dynamic light scattering, and X-ray diffraction. The formed AgNPs had a spherical shape with a particle size range of 11.02–17.92 nm. The antimicrobial activity of AgNPs was investigated in vitro and in vivo against *S. aureus* clinical isolates. The minimum inhibitory concentration (MIC) of AgNPs ranged from 4 to 64 µg/mL. AgNPs significantly decreased the membrane integrity of 45.8% of the isolates and reduced the membrane potential by flow cytometry. AgNPs resulted in morphological changes observed by SEM. Furthermore, qRT-PCR was utilized to examine the effect of AgNPs on the gene expression of the efflux pump genes *nor*A, *nor*B, and *nor*C. The in vivo examination was performed on wounds infected with *S. aureus* bacteria in rats. AgNPs resulted in epidermis regeneration and reduction in the infiltration of inflammatory cells. Thus, GTLE could be a vital source for the production of AgNPs, which exhibited promising in vivo and in vitro antibacterial activity against *S. aureus* bacteria.

**Keywords:** AgNPs; antioxidant activity; flow cytometry; *Gardenia thailandica*; HPLC; infected wound; qRT-PCR

## **1. Introduction**

Nanotechnology is a relatively novel discipline with massive applications, including those in the medical and pharmacological industries [1]. Recently, there is a growing interest in the usage of green-synthesized biocompatible silver nanoparticles (AgNPs) in various applications including antimicrobial products, anti-fungal preparations, drug delivery, the textile industry, and food packaging. Several chemical and physical methods for the synthesis of AgNPs have been reported, including sol-gel, chemical reduction,

**Citation:** Attallah, N.G.M.; Elekhnawy, E.; Negm, W.A.; Hussein, I.A.; Mokhtar, F.A.; Al-Fakhrany, O.M. In Vivo and In Vitro Antimicrobial Activity of Biogenic Silver Nanoparticles against *Staphylococcus aureus* Clinical Isolates. *Pharmaceuticals* **2022**, *15*, 194. https://doi.org/10.3390/ ph15020194

Academic Editor: Fu-Gen Wu

Received: 6 January 2022 Accepted: 1 February 2022 Published: 3 February 2022

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physical vapor deposition, thermal decomposition, and microwave irradiation [2,3]. Unfortunately, these techniques have many drawbacks such as the expense, use of high energy and/or hazardous chemicals, and production of toxic byproducts that are unsafe to the environment [4,5]. Besides, these toxic chemicals are attached to the end products, which considerably limits their application [2,6].

To overcome these limitations, it is crucial to find eco-friendly, easy to use, cost effective, and nontoxic alternative methods for the fabrication of AgNPs [4]. Recently, new methods based on green synthesis are emerging. These methods use eco-friendly compounds as reducing agents [1]. Plants and microorganisms are considered nontoxic biological reproducible resources that are safe for humans and the environment. They may be more suitable alternatives for the biosynthesis of AgNPs [7]. Plant extract nanoparticles are more favorable than microorganism-based nanoparticles since they do not require particular and complex processes such as culture management, isolation, and several purification steps [8]. In addition, using plants for the synthesis of nanoparticles has other advantages, such as the use of safer solvents, milder response conditions, more feasibility, and their various uses in surgical and pharmaceutical applications [3]. Due to the aforementioned limitations, researchers have developed green methods that employ various plant parts such as the leaf, peel, flower, fruit, and root. Numerous plant extract compounds (e.g., ascorbic acids, flavonoids, polyphenols, proteins, and terpenoids) play important roles in metal ion uptake, precursor salt reduction, and capping agents. Furthermore, several of them have antibacterial capabilities [9].

*Gardenia thailandica* Triveng. is a flowering plant native to Thailand. Gardenia species have a high medicinal potential and a long history of usage in traditional medicine to cure a variety of ailments such as jaundice, fever, hypertension, and skin ulcers. In addition, various Gardenia species have been linked to a variety of pharmacological effects, including anti-inflammatory, anti-viral, anti-cancer, and anti-apoptotic properties [10–13]. One of the most potential antimicrobials used in nanomedicine are AgNPs. AgNPs can interact with a microorganism's cell wall, producing reactive oxygen species that eventually cause cell death [9]. As a result, we can speculate that using *G. thailandica* extract will produce AgNPs with improved antimicrobial activity.

*Staphylococcus aureus* is a highly virulent pathogenic bacteria that can cause various clinical infections in humans. They are a major cause of infective endocarditis and bacteremia, in addition to skin and soft tissue, osteoarticular, pleuropulmonary, and device-associated infections [14]. Besides the various virulence factors they possess, antimicrobial resistance is widely spreading among these bacteria. Thus, new approaches should be studied to overcome different infections caused by *S. aureus* [15]. The green synthesized AgNPs could be a therapeutic alternative to the currently present antimicrobials.

In this study, we aimed to green synthesize AgNPs from *Gardenia thailandica* leaf extract (GTLE). Then, the produced AgNPs were characterized by different techniques. Furthermore, the antibacterial activity of the synthesized AgNPs was studied both in vitro and in vivo against *S. aureus* clinical isolates.

#### **2. Results**

#### *2.1. High Performance Liquid Chromatographic Coupled with Diode Array Detector (HPLC-DAD) Analysis*

The identification and quantification of phenolic compounds of GTLE was performed using the HPLC-DAD. Figure 1 displays the HPLC-DAD chromatogram for the identified flavonoids and phenolic compounds of GTLE. The abundant phenolic compounds were chlorogenic acid (1441.03 µg/g), while the major identified flavonoid compound was quercetin-3-rutinoside (2477.37 µg/g), as shown in Table 1.

**Figure 1.** HPLC-DAD of GTLE (320 nm). Chl—chlorogenic acid; syr—syringic acid; van—vanillic acid; *p-*co—*p-*coumaric acid; q-rut—quercetin rutinoside; ros—rosmarinic acid; cin—cinnamic acid; api—apigenin; lut—luteolin; chr—chrysin.

**Table 1.** Chemical composition analysis of the phenolic and flavonoid compounds of GTLE by HPLC-DAD.


\* ND stands for none detected.

*2.2. Characterization of the Green-Synthesized AgNPs*

2.2.1. Physical Observation

After 3 h of preservation in a cool and dark area, the physical appearance of the AgNO<sup>3</sup> solution changed to a dark solution after the addition of GTLE, indicating the chemical reduction reaction and synthesis of AgNPs.

#### 2.2.2. UV-Vis Spectroscopy

UV-Vis spectroscopy was utilized as the first proof of nanoparticle formation owing to the selectivity of UV towards the formed nanoparticles. Since AgNPs have a characteristic optical reflectivity, they interact strongly with specific wavelengths of light. Because of the collective oscillation of electrons in AgNPs, free electrons produce a surface plasmon resonance (SPR) absorption band [16]. The absorption of AgNPs is controlled by the dielectric medium, chemical environment, shape of the particles, and particle size. The UV measurements of the produced AgNPs had an absorbance at 418 nm (Figure 2).

**Figure 2.** UV spectrum of the biosynthesized AgNPs by GTLE compared to GTLE.

2.2.3. Fourier-Transform Infrared (FTIR) Spectroscopy

The identity of the functional chemical groups of the GTLE involved in the reduction reaction to produce AgNPs was configurated by FTIR spectroscopy measurements. Peaks at 3417, 2926, 1632 cm−<sup>1</sup> represent the functional groups as follows; OH, C aliphatic, and C=O of phenolic acids and flavonoids, while the polyphenols and aromatic compounds were represented by the peak at 1453 cm−<sup>1</sup> . The secondary OH groups of GTLE were confirmed by the peak at 1080 cm−<sup>1</sup> (Figure 3).

**Figure 3.** FTIR spectrum of the biosynthesized AgNPs by GTLE compared to GTLE.

2.2.4. High-Resolution Transmission Electron Microscope (HR-TEM)

The green-synthesized AgNPs using GTLE as a reducing agent were examined using HR-TEM, which revealed the formation of spherical shaped AgNPs with a particle size range of 11.02–17.92 nm and an average size of 14.24 nm (Figure 4). In addition, the selected area electron diffraction (SAED) pattern confirmed the crystalline nature of the formed AgNPs.

**Figure 4.** HR-TEM micrographs of the biosynthesized AgNPs using GTLE; (**A**): at 20 nm, (**B**); at 50 nm, (**C**): SAED confirmed the crystalline nature of the formed AgNPs.

2.2.5. Zeta Potential and Dynamic Light Scattering (DLS)

We used the zeta potential technique to evaluate the surface charge of the greensynthesized AgNPs. Herein, AgNPs had a zeta potential value of −6.54 ± 0.6 mV, where the negative charge highlighted the stability of the formed nanoparticles (Figure 5A).

**Figure 5.** Zeta potential analysis (**A**) and DLS (**B**) of the biosynthesized AgNPs by GTLE.

Legend shells including the metallic shell of the formed nanoparticles were measured using the DLS technique; they had a size of 77.4 ± 1.88 nm (Figure 5B).

2.2.6. X-ray Diffraction (XRD)

The intense peaks were noticed at the 2θ scale of 38.26, 44.47, 64.71, and 77.73 corresponding to the (111), (200), (220), and (311) planes for silver, respectively (Figure 6).

**Figure 6.** X-ray diffraction pattern of the biosynthesized AgNPs using GTLE.

2.2.7. Scanning Electron Microscope (SEM)

SEM is a useful tool for investigating an object's surface images. It can precisely illustrate the particle size, shape, and distribution of the tested material. In addition, it can determine the morphological appearance of the studied object and determine whether its size is at the micro- or nanoscale. SEM analysis of the biosynthesized AgNPs revealed that they are spherical in shape with a tendency to aggregate (Figure 7).

**Figure 7.** SEM of AgNPs biosynthesized using GTLE.

#### *2.3. Total Content of Flavonoids and Polyphenolics*

Total flavonoids were found to have a content of 162.98 mg/g equivalent to quercetin, while total polyphenols had a content of 287.89 mg/g equivalent to gallic acid. Findings indicate that *G. thailandica* possesses high contents of polyphenols and flavonoids.

#### *2.4. Antioxidant Activity*

The antioxidant activity of GTLE was investigated in this study using radical scavenging and metal-reducing assays. Radical scavenging assays used were 2,2-diphenyl-1 picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) tests. GTLE exhibited antioxidant activity by DPPH and ABTS as (IC<sup>50</sup> 72.91 µg/mL) and (211.60 mg Trolox equivalents (TE)/mg), respectively. The metal-reducing assay used was the ferric reducing antioxidant power assay (FRAP) and the activity of GTLE was 70.95 mg TE/mg.
