Cytotoxic Activity and Antibiofilm Efficacy of Biosynthesized Silver Nanoparticles against Methicillin-Resistant Staphylococcus aureus Strains Colonizing Cell Phones

The interest for green synthesis of metallic nanoparticles (NPs) has acquired particular attention due to its low toxicity and economic feasibility compared with chemical or physical process. Here we carried out an extracellular synthesis approach of silver nanoparticles (AgNPs) using dried orange peel extract. Characterization studies revealed the synthesis of 25–30 nm AgNPs with distinct morphology as observed in transmission electron microscopes. Dynamic light scattering spectroscopy and Fourier transform infrared spectroscopy analyses further characterized nanoparticles confirming their stability and the presence of functional groups. The biological properties of biosynthesized AgNPs were subsequently investigated. Our results revealed anticancer activity of biogenic silver NPs against the B16 melanoma cell line with an IC50 value of 25 µg/ml. Additionally, the developed AgNPs displayed a considerable antagonistic activity against methicillin-resistant Staphylococcus aureus (MRSA) strains colonizing cell phones, with inhibition zones between 12 and 14 mm and minimum inhibitory concentration values between 1.56 and 12.5 µg/ml. Furthermore, the AgNPs exhibited potent antibiofilm activity against MRSA strains with the percent biofilm disruption reaching 80%. Our results highlighted the efficacy of biosynthesized AgNPs against bacterial biofilms and pointed to the exploration of orange peels as a natural and cost-effective strategy.


Introduction
Antimicrobial resistance is still one of the major problems threatening human health. It increases mortality and leads to longer hospital stays and greater medical costs [1]. Bacterial biofilm formation represents one of the most serious health hazards and leads to severe contamination problems. is microbial structure is difficult to combat since it has been revealed that antibiotic resistance is due to bacteria existing within a biofilm but rather to free bacteria [2]. e complexity of the bacterial biofilm structure mediated the inactivation or modification of antimicrobial enzymes and enabled the antimicrobial enzymes to penetrate the biofilm [3].
Staphylococcus aureus is one of the opportunistic pathogens often associated with serious nosocomial infections. Its virulence factors along with its multidrug resistance represent a major threat worldwide [4]. S. aureus is well known for its biofilm-forming ability involving various virulence factors such as bacterial surface proteins, fibronectin-binding proteins, the autolysin AtlA, protein A, biofilm-associated protein Bap, phenol-soluble modulins, proteases, nucleases, teichoic acids, the polysaccharide intercellular adhesin, and extracellular DNA [5]. Biofilmforming capacity along with antibiotic resistance is crucial for the success of S. aureus as a dreaded pathogen in both healthcare and community settings. Moreover, methicillinresistant S. aureus (MRSA) strains are usually resistant to commonly used antibiotics [6].
Nanotechnology is an emerging area of science dealing with nanoparticles of various materials.
Eco-friendly synthesis of silver nanoparticles can be used as a promising approach for multifunctional applications [7]. Hence, effective approaches for green synthesis of metallic NPs using natural resources such as plants, algae, bacteria, and fungi were largely reported [8]. Interestingly, plant extract-functionalized AgNPs have drawn considerable attention due to their exclusive biological properties, low toxicity, and good stability [9,10]. e specific characteristics of nanoparticles such as their small size, various morphologies, and distribution improve their functional properties such as antioxidant, antiproliferative, antibacterial, and antibiofilm activities [7,11]. e effectiveness of nanoparticles against drug-resistant bacteria as well as their virulence factors encourages further exploration in this field of research [12]. Silver nanoparticles obtained by green synthesis are largely investigated against various pathogenic microorganisms [13]. ey are effectively used in the medical field as antimicrobial agents against active bacteria, viruses, and fungi [14]. e high solubility and the small size of nanoparticles represent specific characteristics to cross tissue barriers of drug-resistant bacteria. Such properties give this nanomaterial a great potential to reduce or limit the development of new mechanisms of resistance against them [15].
In this regard, we investigate the biosynthesis of AgNPs using dried orange peel extract, and subsequently, we evaluate its potentiality as antibacterial and antibiofilm agents against MRSA strains.

Preparation of the Extract.
e dried peel of an orange (Citrus sinensis) was collected and cut into small pieces of approximately 1 cm. Peel pieces were then placed in a 250 mL conical flask containing 100 mL of double distilled water. After boiling for 30 min, the mixture was filtered to remove coarse pieces of peel and the sample was stored at 4°C before being used for the synthesis of AgNPs.

Biosynthesis of AgNPs.
An aqueous solution of silver nitrate (AgNO 3 ; Scharlab, Spain) was prepared and used for the synthesis of silver nanoparticles. Briefly, 10 mL of the prepared extract was added to 250 mL conical flasks containing 100 mL of AgNO 3 (1 mM) and stirred continuously (200 tr/min) at 80°C for 90 and 180 min. e color change of the mixture to brown indicates the formation of AgNPs. Once the process of biosynthesis was completed, nanoparticles were retained by ultracentrifugation at 14,000 rpm for 30 min, washed, and suspended in deionized water.

Characterization of Synthesized AgNPs.
e reduction of silver ions into silver particles was monitored by UV-visible spectroscopy (Evolution 60, ermo Scientific) at a wavelength of 300-800 nm. A dynamic light scattering spectroscopy (DLS) analysis was conducted to measure the particle size of the prepared AgNPs in an aqueous solution (hydrodynamic diameter) and to determine the polydispersity index (PDI) and zeta potential (surface charge) using a Zetasizer Nano-S (Malvern ® instruments, UK) at 25°C. In order to avoid multiple scattering effects, the AgNPs were diluted 20-to 200-folds with deionized water. All measurements were undertaken in triplicate (n � 3) and results were expressed as mean ± SD. Fourier-transform infrared (FTIR) spectra obtained using an FTIR spectrophotometer (Perkin Elmer Spectrum FTIR Spectrometer, USA) at wavenumbers between 450 and 4000 cm −1 were conducted to determine the role of the phytoconstituents in NP synthesis. e AgNPs size was determined using transmission electron microscopy (TEM). High-resolution transmission electron microscopy (HR-TEM) was performed by using a TALOS F200x instrument ( ermo Fisher Scientific, Waltham, MA, USA). TEM analysis was performed at 200 kV and 5.5 µA. ImageJ software (ImageJ 1.48v) was used to estimate the average particle size distribution.

Cytotoxic Activity of AgNPs.
e cytotoxic effect of the prepared AgNPs was evaluated by the conventional MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) reduction assay against B16 melanoma cell lines [16]. Cells were subcultured in DMEM (Sigma) supplemented with 10% FCS, 1% L-glutamine (200 mM), and 1% of mixture of penicillin (100 IU/ml) and streptomycin (100 IU/ ml), at 37°C with 5% CO 2 . After distribution of 2.5 × 10 4 B16 cells per well in 96-well plates, various concentrations of the AgNPs (viz., 3.125, 6.25, 12.5, 25, 50, and 100 µg/ml) were added before the final incubation at 37°C for 24 h. A negative control with untreated cells was also evaluated. After treatment, the plates were incubated in the MTT solution (a final concentration of 0.5 mg/mL) for 3 h. e darkblue formazan crystals that formed in intact cells were dissolved with DMSO, and the absorbance at 570 nm was measured with a spectrophotometer microplate reader (Bioteck, Elx 800). is colorimetric assay provides measurement of mitochondrial metabolic rate reflecting the viability in treated and untreated cells [17]. Results were expressed as the percentage of MTT reduction relative to the absorbance measured from negative control cells. All assays were performed in triplicate.

Antistaphylococcal Activities
2.5.1. Microorganisms. Determination of the antibacterial and antibiofilm activities of biosynthesized AgNPs was carried out against the reference strain S. aureus ATCC 6538 and two MRSA isolates (68T and 12C) colonizing the mobile phones of Tunisian students. ese strains were previously characterized for their biofilm formation ability [18].

Disk Diffusion
Method. Selected S. aureus strains were initially subcultured on nutrient agar medium. en, the turbidity of an overnight-grown culture was adjusted to an optical density of 0.5 McFarland (McF). Each strain was spread over the Muller Hinton (MH) agar using sterile swabs. Disks of 6 mm diameter were placed on MH agar plate and impregnated with 50 µg of AgNPs diluted in sterile 10% DMSO. e plates were then incubated at 37°C for 24 h and the appeared inhibition zones of the tested bacteria were measured. All assays were performed in triplicate [19].

Determination of Minimum Inhibitory and Minimum Bactericidal Concentrations.
e minimum inhibitory concentration (MIC) values for AgNPs against MRSA strains were determined according to Merghni et al. [19]. e turbidity of an overnight grown culture was adjusted to 0.5 McF standards. e liquid dilution assay in MH broth was determined using a 96-well microtiter plate. e AgNPs were added aseptically to sterile 96-well microtiter plates (190 μl per well) by twofold serial dilutions in Muller Hinton (MH) broth with 10% DMSO. e resultant doses of AgNPs in each solution (ranged between 50 µg/ml and 0.09 µg/ml) and the inocula (10 μl) of each strain were added to each well. Wells containing only bacteria without AgNPs were considered as positive control. Negative control consists of wells made of sterile medium. Treated microplates were then incubated at 37°C for 24 h and the MIC was defined as the lowest concentration of the samples (AgNPs) that inhibited the bacterial growth.
To determine the minimum bactericidal concentration (MBC) values, a volume of 20 μL from wells without bacterial visible growth was removed and plated on MH agar. Subsequently, the inoculated plates were incubated for 24 h at 37°C and the MBC was determined as the lowest concentration of the samples (AgNPs) that killed 99% of the tested bacteria [19].

Inhibition of Bacterial Cell Attachment by AgNPs.
e antiadhesion properties of AgNPs against MRSA strains were tested following a microplate biofilm assay [20]. Overnight cultures grown in BHI broth were diluted to 10 6 cfu/ml in BHI supplemented with 2% glucose (w/v). A 100 μl aliquot for each strain was transferred to a 96-well microtiter plate, followed by the addition of 100 μl of a subinhibitory concentration (1/16 to 1 × MIC) of the AgNPs dissolved in sterile BHI with 10% DMSO. e plates were kept undisturbed for 24 h at 37°C. Following incubation, the culture supernatant was discarded, and crystal violet (CV)stained biofilm cells were determined at 570 nm using a microplate reader (D.E.E.D Reader, Bio-Rad Instruments). e percentage of inhibition of cell attachment was obtained by the following formula [20]:

Reduction of Biofilm Growth and Development by AgNPs.
Biofilms of S. aureus strains were allowed to develop for 48 h at 37°C in a 96-well microtiter plate, followed by the addition of the AgNPs. 100 μl of the tested agent was dissolved in BHI supplemented with 10% DMSO to yield a range of concentrations of 1 × MIC, 2 × MIC, and 4 × MIC per well. e plates were further incubated for 24 h followed by an assessment of biofilm biomass by CV staining. CV-stained biofilm cells were quantified at 570 nm with the microplate reader, and the percentage of biofilm eradication was obtained by the following formula [20]: 2.9. Statistical Analysis. All data were expressed as mean-± standard deviation (SD) from three independent experiments. Statistical analysis was performed with STATGRAPHICS Centurion XV, version 15.2.11 (StatPoint, Inc.). Differences between sample groups were analyzed using multiple-way analysis of variance (ANOVA) followed by Tukey's post hoc test. p value of <0.05 was considered significant.

Characterization of Biosynthesized AgNPs.
After addition of the dried orange peels extract to 1 mM of AgNO 3 , the color of the reaction mixture started changing to yellowish brown within 10 min. e final color (dark brown) of the solution deepened with an increase in time (Figure 1). One of the first indications of the biosynthesis of silver nanoparticles was represented by the color change of the reaction mixture as a visual marker [21]. e color transition reveals the biotransformation of the Ag + ion into Ag0 which indicates the biosynthesis of AgNPs [22,23]. It was previously reported that the appearance of yellowish brown color in the reaction mixture of AgNPs solution was due to the excitation of surface plasmon vibrations [24]. Apart from the color change of the full reaction substrates, the formation of AgNPs using aqueous extracts from dried orange peels and AgNO 3 (1 mM) solution was confirmed using ultravioletvisible spectroscopy. Free electrons from metal nanoparticles such as silver and gold give rise to surface plasmon resonance (SPR) absorption band [25]. e characteristic SPR band of biogenic AgNPs occurs at 426 nm for reactions carried out at 80°C (Figure 1) indicating the reduction in the particle size at 80°C. e nucleation and growth mechanism affect nanoparticle formation. Particularly, smaller particle sizes were observed and a higher number of nuclei were formed at higher temperature [26]. e results of DLS of biosynthesized AgNPs using dried orange peel extract are shown in Table 1. e hydrodynamic diameter (nm) of biosynthesized AgNPs was 189.3 ± 30.19. Additionally, the zeta potential of the produced nanoparticles was −25.6 ± 0.08 (Table 1), indicating their stability. In fact, it was reported that a negative value of zeta potential indicates electrostatic repulsion among the particles, thereby increasing the stability of the formulation [27,28]. e PDI was found to be 0.236 ± 0.04 (<0.3), reflecting a homogeneous and stable dispersion of the droplet size [29]. e transmission electron micrograph of the biosynthesized nanoparticles ( Figure 2) showed the lack of agglomeration signs which confirmed their stabilization. e average size of these AgNPs was found to be 27.5 nm with a size range of 10 to 50 nm (Figure 2(c)). e particles with sizes 25-30 nm were the predominant, representing 25% of the total content. In general, the NPs were spherical, monodispersed, and uniformly distributed. Interestingly, monodispersity and stability are important and desired characteristics for commercial application of NPs [30]. e functional groups present in synthesized silver nanoparticles were determined by FTIR analysis (Figure 3). In the range of 3500 to 3400 cm −1 , chemical groups corresponding to hydroxyl stretching (-OH) are noticeable in the structure. e double peaks at 1633 cm −1 point out the stretching vibration of the C�O group, suggesting the presence of carbonyl of nonsubstituted amide and water [31]. e existence of specific chemical groups in both solutions confirms the role of orange peel extract as a capping and reducing agent in the process of AgNPs biosynthesis. ereby, the described phytochemical groups are implicated in the synthesis of nanoparticles through their interaction with metal salts [32].

Cytotoxic Activity of AgNPs.
e cytotoxic effect of the synthesized AgNPs was evaluated against the B16 melanoma cell line using the colorimetric assay (MTT). Figure 4 shows the different percentages of viability of B16 cells exposed to various concentrations of AgNPs. Except for the concentration of 3.12 µg/ml of AgNPs, other increased concentrations showed significant effects when compared to untreated cells (p < 0.05). e IC50 value were determined to be 25 µg/ml. Another study reported the anticancer activity against the B16 melanoma cell line of biosynthesized gold nanoparticles using Siberian ginseng with an IC50 value of 10 µg/ml [33]. e use of silver nanoparticles as a safe strategy for the development of anticancer therapy is of interest since they have remarkable effectiveness against skin   [34][35][36]. Recently, biosynthesized metal NPs have attracted highlighted interest for their anticancer activity against a variety of tumor cells such as lung cancer cells [37], cervical cancer cells [38], and skin cancer cells [34]. Molecular mechanisms of metal NPs-induced cytotoxicity are related to the excessive production of reactive oxygen species (ROS) resulting in direct DNA damage and alteration in the mitochondrial membrane which consequently induces apoptosis and necrosis pathways [39,40].   Table 2. MRSA strains 12C and 68T are sensitive towards the AgNPs with IZs of between 12 and 13.66 ± 0.58 mm (p < 0.05), respectively ( Figure 5). Additionally, AgNPs displayed bacteriostatic effect against the majority of tested strains at a concentration of 12.5 µg/ml. Interestingly, 1.56 µg/ml of AgNPs are able to inhibit the bacterial growth of MRSA strain 12C. e MBC values of the tested agents were found to be similar to those against S. aureus strains (50 µg/ml). e inhibition zones (IZs) found in our study are higher when compared to those reported by Kaviya et al. [26] showing IZs values between 7.8 and 9.2 nm against S. aureus.
e AgNPs in their study were biosynthesized using fresh peels of Citrus sinensis unlike in our study which are dried peels. e superior activity of the AgNPs could be due to their small particle size (25-30 nm), facilitating easy passive transport through the cell membrane of treated bacteria. Our findings are in agreement with previous studies dealing with the biosynthesis of nanoparticles from silver nitrate using waste plant extracts and showing the enhancement of the antibacterial activity of green-synthesized AgNPs [13]. e mechanism through which AgNPs are active against bacteria lies mainly in their capacity to induce cell damage. Previously, the bactericidal effect of silver nanoparticles has been elucidated by various reports. Apart from their interaction with the bacterial cell surface, AgNPs can also cross the cell membrane, reach the cytoplasm [41], and attach to the DNA inhibiting its replication. It can also interact with the bacterial ribosome [42] or damage some enzymes structure, leading to eventual bacterial death [43]. Ample of studies have been performed to estimate the antibacterial potentials of AgNPs and revealed that these nanoparticles trigger oxidative stress, protein dysfunction, membrane and DNA damage, inducing microbial cell damage [44].  S.a 12C S.a 68T S.a 6538 To evaluate the antiadhesion effects of AgNPs, the selected S. aureus strains were cultured in microtiter plates for 24 h in the presence of subinhibitory concentrations of the test agents (1/16× to 1×MIC). Our results showed that AgNPs are active against biofilm of all the tested strains even at a concentration of 1/8 × MIC, corresponding to 0,195 µg/ ml against the 68T strain and 1.56 µg/ml against both 6538 and 12C strains (p < 0.05). With these concentrations, the absorbance at 570 nm was found to be less than 1 reflecting a weak biofilm capability of treated bacteria and subsequently an antiattachment effect which is a crucial phase (step) for biofilm formation and development [19]. AgNPs exerted an antiattachment effect, and they were more active against all tested S. aureus strains as shown in Figure 6. Here we present inhibition of bacterial adhesion at an early phase of biofilm formation. Generally, nanoparticles can interact with microbial biofilm in three steps. e first interaction concerns the transportation of these particles around the biofilm. e second one is their attachment to the surface of bacterial biofilm and finally the migration within this structure [45].  Biofilm formed by selected S. aureus strains were treated with biosynthesized AgNPs tested at various concentrations (CMI, 2 × CMI, and 4 × CMI). Our results showed various effects on the development of preformed biofilms with percent reduction values ranging from 51.18 ± 6.91 to 91.47 ± 4.36 (Figure 7). We noted that the AgNPs were more effective against the S. aureus ATCC 6538 strain known as biofilm-forming bacteria, with percent reduction values exceeding 91% at a concentration of 4 × MIC corresponding to 50 µg/ml (p < 0.05). AgNPs were also highly effective against MRSA 12C strain with the percentage of biofilm eradication reaching 90.07 ± 6.91% (p < 0.05). Previously, the antibiofilm activity of AgNPs against Gram-positive and Gram-negative bacteria such as MRSA, Streptococcus mutans, Acinetobacter baumannii, and Pseudomonas aeruginosa as well as fungi Candida albicans was studied extensively and the potentiality of AgNPs was reported [46,47]. Exposure to AgNPs leads to bacterial cell death due to stable adherence to the bacterial cell wall followed by penetration of the bacterium and rupturing of the cell membrane, reducing its permeability and respiration [41,47].

Conclusion
e present study emphasized the biosynthesis, characterization, and assessment of cytotoxic and antibiofilm effects of silver nanoparticles. e obtained AgNPs from dried orange peel extract showed good stability and anticancer activity and exhibited strong antistaphylococcal and antibiofilm efficacy against tested strains. All the characteristics of these biosynthesized AgNPs incite their application as a potent antibacterial agent against MRSA biofilm as well as to prevent surface contamination and subsequent infections.

Data Availability
All the original data, especially laboratory notebooks, are available on request.

Conflicts of Interest
e authors have no conflicts of interest to declare in this work.