Potential Impact of Reduced Graphene Oxide Incorporated Metal Oxide Nanocomposites as Antimicrobial, and Antibiofilm Agents Against Pathogenic Microbes: Bacterial Protein Leakage Reaction Mechanism

In the current study, nanocomposites-based reduced graphene oxide (RGO) and metal oxides (AgO, NiO, and ZnO) were fabricated. The starting precursor and RGO were characterized by XRD, Raman, SEM, and HRTEM, while SEM and EDX mapping validated the synthesized nanocomposites. In addition, ZOI, MIC, antibiofilm, and growth curve were tested. The antimicrobial reaction mechanism was investigated by protein leakage assay and SEM imaging. Results revealed that all synthesized nanocomposites (RGO-AgO, RGO-NiO, and RGO-ZnO) have outstanding antimicrobial activity against pathogenic bacteria and unicellular fungi. Moreover, RGO-AgO, RGO-NiO, and RGO-ZnO nanocomposites exhibited an antibiofilm activity percentage against Staphylococcus aureus (91.72%), Candida albicans (91.17%), and Escherichia coli (90.36%). The SEM analysis of S. aureus after RGO-AgO treatment indicated morphological differences, including the whole lysis of the outer surface supported by deformations of the bacterial cells. It was observed that the quantity of cellular protein leakage from S. aureus is directly proportional to the concentration of RGO-AgO, RGO-NiO, and RGO-ZnO nanocomposites and found to be 260.25 µg/mL, 110.55 µg/mL, and 99.90 µg/mL, respectively. The prepared nanocomposites promise to treat resistant microbes as a new strategy for managing infectious diseases.


Introduction
Fast development in microbial contamination was increased, especially after the microbial resistance to some synthetic antibiotics [1]. Escherichia coli is distinct bacteria obtained directly from the intestinal region of all individuals and several animals. Staphylococcus aureus is an important pathogen that has a major impact on human health. Although it is notorious for causing skin and softtissue infections, it has the ability to infect nearly every organ system in the human body, often with fatal consequences 2. E. coli was causing enteric infection and other intestinal disorders such as urinary tract infection (UTI) [3]. Biofilms are the predominant routine life of bacteria in all environments, either natural or artificial. Biofilms can progress on a wide variety of surfaces. Biofilms were involved in food spoilage, water pollution, infectious diseases and industries [4]. The ability of S. aureus to produce biofilms were fully connected to the creation of polysaccharide intracellular adhesion (PIA) and adhesions called microbial surface components recognizing adhesive molecules matrix (MSCRAMM) which become involved as primary agents in biofilm formation [5]. In the current age, the utilization of inorganic antimicrobial agents has brought much attention to the control of pathogenic microorganisms [6]. The distinct advantages of inorganic antimicrobial composites, as related to their organic counterparts, are safer and more stable during high temperatures [6]. Therefore, the searching or synthesis of new antimicrobial agents is required. Recently, graphene-based materials (such as graphene, graphene oxide (GO), reduced GO (RGO)) have attracted a lot of research attention [7][8][9]. Graphene oxide (GO) is the oxidized derivative of a graphene molecule, obtained by acid oxidation of graphite, chemical or thermal reduction, resulting in a spin-off of GO called RGO [10,11]. Previous studies reported that RGO has antimicrobial activity [12][13][14], and GO when conjugated with active ingredients possesses antimicrobial and anti-nematodal activities [15,16].
Herein, RGO based-metal oxide nanocomposites have much attention as antimicrobials in last year [17][18][19][20]. Similar studies explain the role of RGO-metal oxide nanoparticles as promising antimicrobial agent, Umekar et al. [21] adopt and mention the bioinspired methods to create the functional RGO and RGO-metal, and metal oxide nanocomposite and summarizes the prevailing state of knowledge regarding the different bioinspired approaches happened to get RGO and RGO-metal, and metal oxide nanocomposite, and their photocatalytic, antimicrobial, and cytotoxic evaluations. Chaudhary et al. [22], specifying the different green and chemical methods for creating RGO and RGO-metal, and metal oxide nanocomposite (RGO-ZnO, RGO-Ag, RGO-MgO). They possess an encouraging property, helping in the significant applications in industry, environment, agriculture, and biomedical fields as antimicrobial agents to fight the pathogenic microbes and as photo-catalysts to remove hazardous heavy metals and dyes. Finally, Potbhare et al. [23], synthesized novel graphene-based silver nanoparticles by a bacterial filtrate as a green method. The synthesized graphene-based silver nanoparticles were characterized totally to ensure the property of the synthesized graphene-based silver nanoparticles. The synthesized nanocomposites were tested against pathogenic bacteria, and the results indicated that they were promised antibacterial agents against the pathogenic E. coli.
Therefore, the current study pointed to synthesize and characterize RGO-based metal oxide nanocomposite (RGO-AgO, RGO-NiO and RGO-ZnO). RGO-metal synthesis was carried out using ascorbic acid as an effective chelating agent using the facile hydrothermal approach.
Ascorbic acid can reduce GO to RGO; in the meantime, it can support the formation and stabilization of ultrafine metal particles on the RGO surface. Therefore enhanced particle dispersion on the substrate surface can be accomplished. The synthesized nanocomposites may find potential applications in industries and medical operation rooms as effective materials to eliminate or reduce pathogenic microbes' dangerous and fetal effects. New methods have been conducted, such as growth curve assay to confirm the qualitative antimicrobial activity and membrane leakage assay to ensure the SEM reaction mechanism. So the novelty to determine the reaction mechanism is in detail.

Materials
Employed chemicals were analytical grade. They were obtained from Sigma-Aldrich and used with no further purification. Natural graphite flakes (99% carbon basis purity) were employed as a precursor for developing reduced graphene oxide (RGO). Potassium permanganate (99%) was utilized as an oxidizing agent; sulfuric acid (98%) and phosphoric acid (85%) were used. Ascorbic acid (99%) was used as a reducing agent. Ethanol (96%) and Acetone (C 99%) were used for all washing steps until the final RGO powder. Silver nitrate, zinc nitrate, nickel nitrate (Sigma Aldrich-USA, 99%) were employed as precursors of metal deposition on the RGO surface. The deposited metal particles were quantitatively converted to the corresponding metal oxide via the calcination process.

Synthesis of Reduced Graphene Oxide
RGO was developed from graphite via two main steps [24][25][26]. The first step includes oxidation and exfoliation of graphite precursors to produce graphene oxide (GO) using the modified hummers method [27][28][29]. In this method, 120 mL of concentrated sulfuric acid (H 2 SO 4 ) and 13 mL of concentrated phosphoric acid (H 3 PO 4 ) (volume ratio *9:1) were mixed. Then 1 gm of graphite was added into the mixing solution under stirring. Potassium permanganate (KMnO 4 ) was added dropwise. The dark violet suspension was treated with 30% H 2 O 2 (50 mL). Upon H 2 O 2 addition, the bright orange color of GO was evolved as schematically presented in Fig. 1. The synthesized GO suspension was washed with de-ionized water. The second step concerns the reduction of GO to RGO. Ascorbic acid was employed as a reducing agent [30,31]; ascorbic acid was added gradually to the heated GO suspension under vigorous agitating until dark black color evolved. Isolated RGO powder was dried at room temperature for 24 h followed by oven drying at 45°C for 24 h.

Synthesis of RGO-Metal Oxide Nanocomposites
RGO-metal oxide hybrid material was prepared via facile solution reduction and co-precipitation method [32]. 10 mg of RGO was dispersed in de-ionized water via sonication. Metal nitrate solution (silver nitrate, zinc nitrate, nickel nitrate; Sigma Aldrich-USA) was added dropwise to the RGO colloid. RGO-metal synthesis was carried out using ascorbic acid as an effective chelating agent using the facile hydrothermal approach. Ascorbic acid can not only reduce GO to RGO, but also it can support the formation and stabilization of ultrafine metal particles on the RGO surface. Therefore enhanced particle dispersion on the substrate surface can be accomplished. The RGO-metal residue was washed with distilled water, centrifuged, and dried. The RGO-metal was quantitatively converted to RGO-metal oxide nanocomposites via calcination at 400°C. The schematic presentation for RGO-metal oxide nanocomposites preparation is represented in Fig. 1.

Characterization Methods
The crystallite sizes and the crystalline structure of starting graphite and developed RGO were determined by the XRD-6000 lists, Shimadzu apparatus, SSI, Japan. The diffracted X-rays' strength was recognized as the diffracted angle 2h. Moreover, Raman spectroscopic measurements were performed on graphite precursor and RGO powder samples using a dispersive Raman microscope (model Senterra II, Bruker, Germany). Spectroscopic analysis was performed on Raman spectra were continuously collected with spectral resolution 4 cm -1 . A Nikon 20 9 objective was used to focus the Raman excitation source (10 mW, 532 nm neodymium-doped yttrium aluminium garnet (Nd: YAG) laser-Bruker, Germany). Morphology of the developed RGO was determined by applying a High-Resolution Transmission Electron Microscope (HRTEM, JEM2100, JEOL, Japan) and Scanning Electron Microscope (SEM, ZEISS, EVO-MA10, Germany). The graphite precursor and RGO-metal oxide nanocomposite surface and morphological features were examined with Scanning Electron Microscope (SEM, ZEISS, EVO-MA10, Germany). Also, EDX spectrum examination (BRUKER, Nano GmbH, D-12489, 410-M, Germany) was used to estimate the elemental composition, purity and relationship of each metal.

Antimicrobial Activity
The antimicrobial potential of as-synthesized RGO-metal oxide nanocomposites against different pathogenic microbes (yeast and bacteria) was examined via employing the agar-disc diffusion method [33]. Firstly, the as-synthesized RGO-metal oxide nanocomposites were dissolved into distilled water with 0.01 mg/mL; 10 ppm. The activity of the as-synthesized compounds was examined against different types of bacteria, namely Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Methicillin-resistant Staphylococcus aureus (MRSA), Proteus vulgaris, Salmonella typhi, and Proteus mirabilis. The examined multi-drug resistance bacteria were tested by VitekÒ two systems (bioMarieux, Marcy-LEtoile, France). Most of them were resistant to antibiotics like Cefapirin, Ciprofloxacin, Amikacin, Norfloxacin, Amoxicillin, Cefoxitin, Gentamicin, Ampicillin, and Cefotaxime. We performed the Biosafety Level-2 (BSL-2) [34]. All the inoculums are Fig. 1 Step-by-step method for reduced graphene oxide (RGO) preparation using modified Hummer's method, and steps of RGOmetal oxide nanocomposite production Potential Impact of Reduced Graphene Oxide Incorporated Metal Oxide Nanocomposites as… 825 established and fixed from 2 to 5 9 10 8 CFU/mL (0.5 McFarland; at 600 nm). The inhibition of the bacterial growth was defined by the zone of inhibition (ZOI) after 24 h of incubation [35]. Additionally, the antifungal potential of the as-synthesized RGO-metal oxide nanocomposites was examined against pathogenic unicellular fungi (Candida albicans and Candida tropicalis).
After that, the inoculums of the tested yeast cells were set from 1 to 4 9 10 8 CFU/mL. Finally, Nystatin (NS) and Amoxicillin (AX) are conducted as standard antibiotics. AX is similar to penicillin in its bactericidal action against susceptible bacteria during the stage of active multiplication. It acts through the inhibition of cell wall biosynthesis that leads to the death of the bacteria [36]. At the same time, NS is an antifungal that is both fungistatic and fungicidal in vitro against many yeasts and yeast-like fungi. It exerts its antifungal effects via the fungal cell membrane [37]. The minimum inhibitory concentration (MIC) of RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites was determined according to the microdilution method in agar diffusion. To determine MIC, different concentrations for each compound (1000-0.5 lg/ mL) were performed. The results were statistically examined by using ONE WAY ANOVA, Duncan's multiple series, and the least significant difference (LSD) that were determined by specific software (SPSS version 15).

Growth Curve Assay
The effect of RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites on the growth of S. aureus was estimated by the growth curve method according to Huang et al. [38]. The bacterial suspension was fixed to 0.5 McFarland (1 9 10 8 CFU/mL) in 5.0 mL of nutrient broth tubes, then 0.5 mL of RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites was inserted individually to each of the tested tubes. The absorbance of the bacterial growth after treatment was assessed every two hour time intervals up to 24 h at a wavelength of 600 nm. The mean of triplicate readings was plotted against the hour intervals to obtain the standard growth curve.

Antibiofilm Potential
Furthermore, a qualitative analysis concerning biofilm restraint was tested as declared by Christensen et al. [39]. The biofilm's definitive study was presented at the tube wall in which the lack and presence of the integrated RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites were confirmed. The antibiofilm of as-synthesized RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites (at 10.0 lg/ mL) was measured against the chosen microbes and was correlated with the control (non-treated one). In summary, 5 mL of the nutrient broth medium was inserted inside all tubes, and the tested bacteria and yeast were inoculated, subsequent adjusted 0.5 McFarland to be 3.5 9 10 8 CFU/ mL. Later, they were incubated at 37.0 ± 0.5°C for 24 h. The media found in control and treated tubes were dropped, mixed with Phosphate Buffer Saline (PBS; pH 7.0), and ultimately preserved. Next, the bacterial and yeast cells that adhered to the tube walls were implanted with 5 mL sodium acetate (3.5%) for approximately 20 min. Finally, they were cleaned with de-ionized water. Biofilms organized inside tubes were stained with 20 mL Crystal Violet (CV; 0.15%) and washed with de-ionized water to eliminate the excess of CV. It must be remarked that, for the semi-quantitative antibiofilm calculation, 5 mL of the absolute ethanol was inserted to separate the stained bacterial and yeast biofilms [40]. UV-Vis. spectrophotometer at 570.0 nm was measured the O.D. of the stained bacterial and yeast biofilms [2]. The bacterial and yeast biofilms inhibition percentage was determined by the subsequent relation (Eq. 1) [41]: Effect of the Synthesized Nanocomposites on Protein Leakage from Bacterial Cell Membranes Pure 18 h bacterial culture was set at 0.5 McFarland (1 9 10 8 CFU/mL), and 100 lL was injected into 10 mL of the nutrient broth, including well-sonicated and dispersed RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites at various concentrations (0.125, 0.25, 0.5, and 1.0, mg/mL). Nanocomposites-free broth injected with culture was used as the control. All the treated samples were incubated at 37°C for 5 h and then centrifuged for 15 min. at 5500 rpm [42]. For the different samples, 100 lL supernatant was combined with 1 mL of Bradford reagent. Optical density had measured at 595 nm after 10 min of dark incubation [42].

Reaction Mechanism Using SEM Imaging
The sensitive microbial cells were washed with PBS three times and eventually fixed with 4.0% glutaraldehyde solution [43]. The preserved microbial cells were regularly cleaned with PBS and repeatedly drained with various ethanol concentrations (30,50,70,90, and 100%) for 15 min at 28 ± 2°C [44]. Next to that, the fixed samples were solidified on an aluminium portion, considering SEM analysis. The morphological characteristics of the control (non-treated microbial cell) and RGO-AgO nanocomposites-treated microbes were observed by SEM examination.

Results and Discussion
Characterizations of Graphite, and Reduced Graphene Oxide (RGO) The graphite precursor was characterized by scanning electron microscopy (SEM). The observed morphological profile of the employed graphite confirmed its flake nature with the lateral dimension within the order of 40-80 lm, as shown in Fig. 2a. In addition, the SEM micrographs demonstrated a multiple-layered structure of about 2-4 lm thickness (Fig. 2b). Moreover, SEM micrographs of synthesized RGO demonstrated the successful exfoliation of graphite to RGO, as presented in Fig. 2c. The obtained RGO sheets appear randomly aggregated thin sheets with distinct edges, wrinkled surfaces, and folding. Moreover, the HRTEM micrograph of RGO (Fig. 2d) confirms the apparent effective exfoliation that the RGO sheets may have consisted of a few layers stacked on top of each other with fewer wrinkles and folding. Both SEM and HRTEM show no residual reactants or byproducts on the RGO surface. Figure 2e represents the acquired Raman spectrum of developed RGO concerning starting graphite (Fig. 2f). Raman spectrum of RGO exhibited G band at 1582 cm -1 (typical also for graphite), and broad D band (characteristic for RGO) appears at 1350 cm -1 ; this characteristic peak confirmed the formation of RGO [45][46][47][48][49].
The XRD diffractogram of starting graphite precursor, and developed RGO is shown in Fig. 2g, h, respectively. The appearance of the (002) with a broad and low-intensity XRD peak centered at 2h & 26.5°(d spacing of 3.380 Å ) verified the successive formation of fragile RGO layers due to high degree of exfoliation few-layer RGO (Fig. 2h) [50,51].
According to the obtained results from SEM, HRTEM, Raman, and XRD (Fig. 2); it is evident that RGO lost the original graphite structure [47]. Reduction of GO leaves aggregated and randomly packed RGO sheets. The obtained Raman spectra confirm the successful preparation of few-layer reduced graphene oxide with typical thicknesses \ 10 nm [45,48,49].

Characterization of RGO-Metal Oxide Nanocomposites
XRD investigate the crystal composition and the usual crystal size of the integrated metal oxide NPs across the RGO, as it outlines the state of the identified crystals [52][53][54]. XRD crystalline structure was presented in Fig. S1 (Supplementary Fig. 1) for the synthesized RGOmetal oxide nanocomposites. The lattice structure and the diffraction design showed a crystalline composition of the loaded metal oxides (NiO, AgO, and ZnO NPs), which clears defects and any interfering materials [55].
The XRD data for RGO-NiO nanocomposites confirmed the composition of the diffraction characteristics dominates 2h for NiO NPs with the corresponding Bragg's reflections at 37.21° (111) [61]. The integrated metal oxides showed a large property regarding the crystalline formation, and the XRD was matched with the recently published articles [62][63][64].
Typical SEM was used to determine the appearance of the prepared nanocomposite and the external morphology [65,66], while the EDX analysis is an analytic technique applied for the elemental study or the chemical characterization of the fabricated samples [52,53,[67][68][69]. SEM micrograph demonstrated the uniform distribution of metal oxide on the RGO surface, as represented in Fig. 3. As shown, the AgO NPs appear as bright spherical particles on the surface of the synthesized RGO sheets as inhibited in Fig. 3a, which confirms the uniform distribution of AgO on the outside of the produced RGO layers. The EDX analysis of the synthesized RGO-AgO nanocomposite is presented in (Fig. 3b), the EDX spectrum recorded that the incorporated RGO-AgO nanocomposite is stoichiometric and similar to a normal composition. The characteristic X-ray peaks of C (61.97%; At.%), O (20.20%; At.%), and Ag (17.83%; At.%) atoms are apparent in the EDX of the RGO-AgO nanocomposite.
The same situation was noted in the case of ZnO NPs distributed outside the RGO surface, (Fig. 3c) and the EDX spectrum for the synthesized RGO-ZnO nanocomposites  outstanding application, especially in the biomedical fields [15]. The elemental mapping analysis of the synthesized RGO-metal oxide nanocomposites is displayed in Fig. 4. The images are identified as Ag, C, and O for the synthesized RGO-AgO nanocomposite (Fig. 4a), as Zn, C, and O for the synthesized RGO-ZnO nanocomposite (Fig. 4b), and as Ni, C, and O for the synthesized RGO-NiO nanocomposite (Fig. 4c). Figure 4 confirmed the synthesized nanocomposite in terms of the appearance of the detected atoms (Ag, Zn, Ni, C, and O) as EDX analysis. On the other hand, the elemental mapping images demonstrated uniform element distribution, the noted purity, and no interfering elements were reported [26].

Antimicrobial Activity of the Synthesized RGO-Metal Oxide Nanocomposites
Recently, nanocomposites are widely-used for treating the resistance microbes which produce slim biofilms [70].
Previous studies reported that RGO nanosheets, RGO nanofilms and RGO paper have potential antibacterial activity against spherical and rod-shaped bacteria with low cytotoxicity [71,72]. Therefore, RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites were synthesized in this study. Antimicrobial activity of RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites against different bacterial and fungal strains was evaluated as shown in Table 1 and Fig. 5.
Overall, all designed composites (RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites) exhibited promising antimicrobial activity against all tested bacterial and Candida strains compared to AX/Nyst as standard antimicrobial agents, where RGO-AgO and RGO-NiO nanocomposites were significantly higher than AX/Nyst. Results revealed that RGO-AgO nanocomposite has the highest effect on most tested bacterial and fungal strains among other RGO-NiO and RGO-ZnO nanocomposites. Table 1 illustrates that RGO-AgO nanocomposite (0.1 mg/mL; 10 ppm) had the highest impact on S. aureus among all tested bacterial strains. The inhibition zone was 33.8 mm; additionally, Furthermore, RGO-NiO nanocomposite had antimicrobial activity but lower than RGO-AgO; the highest effect was against E. coli, S. aureus and C. albicans among fungal and bacterial strains with inhibition zones 25.0, 22.2, and 21.5 mm, respectively. On the other hand, the lowest result was towards K. pneumoniae with inhibition zones 8.5 mm, as shown in Table 1 and Fig. 5.
Incorporation of RGO with metal oxide nanoparticles prevents aggregation leading to nanocomposite with high stability and better antibacterial activity than each one alone [73,74]. Sadhukhan et al. [74], reported that RGO/ NiO NCs might be used as antibiotics in the future due to non-toxic, cheap, eco-friendly and highly effective against the pathogenic microorganisms. Also, Hsueh et al. [19],

Growth Curve Assay of RGO-NiO, RGO-AgO, and RGO-ZnO Nanocomposites
The influence of RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites on the majority of S. aureus is presented in Fig. 6. The growth rate of S. aureus in the control sample happened quickly, with the most potent optical density at k = 600 nm (OD 600 ) values had arrived at 3.125 nm. Indifference, the OD 600 values of the synthesized RGO-NiO, RGO-AgO and RGO-ZnO nanocomposites were lower (0.988 nm, 0.3569 nm, and 0.685 nm, respectively), showing the repression impact on the majority of S. aureus. RGO-AgO nanocomposite display further suppressing power more than RGO-NiO, and RGO-ZnO nanocomposite that may be defined by the unique antibacterial activity of AgO NPs that described by previous studies [2,76].
Generally, on the NPs surface, the photo-generation of reactive oxygen species (ROS) has been described by early papers [77,78]. The synthesized RGO-NiO, RGO-AgO and RGO-ZnO nanocomposites form ROS causes protein oxidation, DNA injury, and lipid peroxidation that can destroy the bacteria without harming the other cells [79]. Moreover, the S. aureus membrane possesses a negative charge, while the metal ions liberated from RGO-metal oxides nanocomposite (Ni 2? , Zn 2? , and Ag ? ) own a positive charge. So, they become in direct contact to cut DNA replication, protein denaturation, and destruction of bacterial cells [80]. The higher hypersensitivity of the Grampositive bacteria to the nanocomposites may be defined as a consequence of the lower stiffness of the bacterial cell membrane [81]. A further possible cause can be the size, appearance, and surface charge of the synthesized RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites, which could afford them more beneficial to connect with Grampositive bacteria. Xu et al. [82], noted that NPs, after UVirradiation for 80 min, cracked the cell membrane of E. coli, indicating that disinfection was performed. Most NPs, and nanocomposites showed antibacterial potential toward different bacterial strains, like S. aureus, P. aeruginosa, and E. coli, and against a broad range of pathogenic unicellular fungi [52,53,70,[83][84][85][86][87][88].

Antibiofilm Potential of the Synthesized RGO-Metal Oxide Nanocomposites
The biofilm development in pathogenic bacteria is identified by exo-polysaccharide production [2]. The tube design was used to define the antibiofilm behavior of the integrated RGO-AgO nanocomposite toward some pathogenic bacteria and unicellular fungi [89].
Our results point to the antibiofilm activity of the RGO-AgO nanocomposite (the most efficient RGO-metal oxide nanocomposite) for S. aureus (an example of sensitive pathogenic bacteria). The complete steps are: (I) Regular microbial growth and reproduction of the distinguished ring in the absence of RGO-AgO nanocomposite and the restraint of the microbial growth in the presence of RGO-AgO nanocomposite, (II) The possibility of staining of the established biofilm with Crystal Violet (CV), which is a qualitative measurement system, and (III) Removal and separating the adhered microbial cells following ethanol reaction for the semi-quantitative evaluation of the biofilm inhibition % (  Results obtained from the tube design had been used to determine the antibiofilm potential of RGO-AgO nanocomposite against S. aureus, which creates a thick whitish-yellow layer in the air-liquid interface in the lake of the RGO-AgO nanocomposite (control). The produced matt layers were fully adhered across the walls of the designed tubes and developed as a blue color following the staining with CV. Next, a dark blue color was created in the produced solution, subsequent dissolving CV with absolute ethanol.
On the other side, in the tubes including S. aureus cells and RGO-AgO nanocomposite (10 lg/mL), a remarkable negative impact was seen as the cells of the tested bacteria did not produce biofilm layers the ring construction was blocked. Also, the adherent cell color was soft, and the blue color was faintly developed following ethanol addition.
A UV-Visible spectrophotometer examined the semiquantitative measurement of the repression percentage (%). The optical density (O.D.) was measured at 570 nm following terminating CV-stained biofilms, which were recognized as a power of their production [89]. Table 2 illustrates the inhibition % after adding 10.0 lg/ mL RGO-AgO nanocomposite, showing that the highest percentage for S. aureus is 91.72%, for C. albicans is 91.17%, and for E. coli is 90.36%. Note that RGO-AgO nanocomposite could manage the biofilm extension at its adhesion strength, which is the initial start in the antimicrobial means [90]. The difference in the hindrance percentage may be linked to several constituents like the great potential of the antimicrobial factors to be connected to the surface due to the enhanced surface area of the integrated RGO-AgO nanocomposite and their particle size, as well as the attack mode and various chemical properties affecting the association and interaction of RGO-AgO nanocomposite among biofilms-producing bacteria [89,91,92]. Figure 7 presents a reviewed diagram concerning the antibiofilm activity of RGO-metal oxide nanocomposites (as inhibition %) toward various pathogenic microbes.

Determination of Protein Leakage from Bacterial Cell Membranes
The quantities of protein discharged in the suspension of the treated S. aureus cells were determined by applying the Bradford method [93]. From Fig. 8, it was observed that the quantity of cellular protein discharged from S. aureus is directly proportional to the concentration of RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites and found to be 260.25 lg/mL, 110.55 lg/mL, and 99.90 lg/ mL after the treatment with 1.0 mg/mL of the tested RGO-AgO, RGO-NiO, and RGO-ZnO nanocomposites, respectively, which proves the antibacterial characteristics of the synthesized nanocomposites, and explains the creation of holes in the cell membrane of S. aureus producing in the oozing out of the proteins from the S. aureus cytoplasm.
These test outcomes revealed that RGO-AgO (the most active nanocomposite) improved the permeability of S. aureus cell membranes more than RGO-NiO, and RGO-ZnO nanocomposites. So it could be assumed that confusion of membranous permeability would be a vital portion of the repression of bacterial mass. Related studies [94], and [95] describe comparable outcomes when incorporated Values are means ± SD (n = 3). Data within the groups are analyzed using one-way analysis of variance (ANOVA) followed by Paul et al. [96] proved that the difference in bacterial cell membrane permeability was shown in percentage difference in corresponding electric conductivity. It was reported that the percentage of relative electric conductivities of all tested samples improves with the rise in the concentration of the treated nanocomposites. The integrity of the bacterial cell membrane had defined by analysis of the discharge of cell components of the bacteria such as proteins; the leakage developed with time as there was constant cell membrane injury that pointed to the leakage of cell components driving to cell destruction [96].

Reaction Mechanism Determination by SEM Imaging
SEM imaging analysis had been conducted to explain the potential antimicrobial mechanism against S. aureus, see Fig. 9. The SEM examination regarding the control bacterial cells in the absence of RGO-AgO nanocomposite (an example of the most potent RGO-metal oxide nanocomposites) conferred bacterial colonies regularly extended with standard normal surface semi-formed biofilm, Fig. 9a. After RGO-AgO nanocomposite treatment, remarkable morphological abnormalities were recognized in S. aureus (Fig. 9b), including the total lysis of the exterior surface supported by deformations of the S. aureus cells. Additionally, the RGO-AgO nanocomposite created the complete lysis of the bacterial cell and cell distortion, which explains the membrane leakage assay and is confirmed by the results obtained (Fig. 8). Eventually, RGO-AgO nanocomposite produced the lysis of the bacterial cell and cell deformity with the drop in the complete viable count, and finally, the biofilm mass was controlled (Fig. 9b).
The schematic illustration in Fig. 10 shows the potential antimicrobial mechanism. There were superior mechanisms like Reactive Oxygen Species (ROS) distribution (superoxide anion; O 2 -) [97], the succession of metal-oxide NPs (NiO, AgO, and ZnO; after being displaced from RGO surface) inside the pathogenic microbes [98], and an alkaline tendency was admitted to show the antimicrobial action mechanism [99]. It is recommended that; metaloxide NPs could change the microbial morphology and their biofilm formation, reduce the microbial membrane permeability and provide the residence of oxidative stress  [97,100]. We understand that RGO-metal oxide nanocomposites begin their performance by wrapping and adhesion at the exterior surface of the microbial cell, producing membrane destruction and changed transport potential [86]. Then, the distribution of metal oxide NPs (NiO, AgO, and ZnO) inside the microbial cell divides all intracellular constructions like plasmid, DNA, and another essential organelle. Ultimately, cellular toxicity happens due to the oxidative stress created by ROS generation. Lastly, at acidic medium, the ionic species were created (Ni 2? , Ag ? , and Zn 2? ions), which made cellular and genotoxicity due to the interaction among the negatively charged vital organs [53,86]. Finally, a recent comparison of the synthesized nanocomposites-based reduced graphene oxide and metal oxides with those reported previously to elaborate and compare the antimicrobial activity and the synthetic method and show why the synthesized nanocomposite is the best is listed in Table 3.

Conclusion and Future Perspective
RGO and its composites with metal oxide (NiO, AgO, and ZnO) NPs were designed and fabricated in the present work. SEM/EDX mapping technique distinguished the effect of the hybridization of metal oxide NPs on the external shape of the prepared RGO. New methods have been conducted, such as growth curve assay to confirm the qualitative antimicrobial activity and membrane leakage assay to ensure the SEM reaction mechanism. So the novelty to determine the reaction mechanism is in detail. The RGO-AgO nanocomposite was more potent in its antimicrobial capabilities than RGO-NiO and RGO-ZnO nanocomposites. It was active even at low concentrations against some examined pathogenic microbes. The results confirmed that RGO-metal oxides nanocomposites    [108] Potential Impact of Reduced Graphene Oxide Incorporated Metal Oxide Nanocomposites as… 837 were so functional against all tested pathogenic microbes due to metal oxides NPs that help interrupt the microbial pathogens' external membrane. The reaction mechanism was studied by membrane leakage assay and SEM analysis because of the oxidative and membrane stress and wrapping isolation due to RGO. In-vitro ZOI results proved that RGO-AgO nanocomposite at concentration 0.1 mg/mL had good antimicrobial activity against E. coli, P. aeruginosa, S. aureus (MRSA), K. pneumoniae, P. vulgaris, S. typhi, P. mirabilis, and C. tropicalis with inhibition zones 31.5, 20.9, 12.8, 10.5, 18.4, 13.3, 20.7, and 21.1 mm, respectively. The growth curve assay of S. aureus in the control sample had been evaluated, with the most potent optical density at k = 600 nm (OD 600 ) values had arrived at 3.125 nm. Indifference, the OD 600 values of the synthesized RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites were lower (0.988 nm, 0.3569 nm, and 0.685 nm, respectively), showing the inhibition impact on the growth of S. aureus. It was observed that the quantity of cellular protein leakage from S. aureus is directly proportional to the concentration of RGO-NiO, RGO-AgO, and RGO-ZnO nanocomposites and was found to be 260.25 lg/mL, 110.55 lg/mL, and 99.90 lg/mL after the treatment with 1.0 mg/mL of the tested RGO-AgO, RGO-NiO, and RGO-ZnO nanocomposites, respectively, which proves the antibacterial characteristics of the synthesized nanocomposites and explains the creation of holes in the cell membrane of S. aureus producing in the oozing out of the proteins from the S. aureus cytoplasm. Finally, it is suggested that the synthesized RGO-metal oxides nanocomposites can be applied in the biomedical treatment due to an encouraged antimicrobial activity but in the limited purposes (the toxicity level must be examined) such as in painting in the operating rooms, face masks, cosmetics, and wound dressing as an excellent antimicrobial agent.

Declarations
Conflict of interest The authors declare that they have no conflict of interest.
Research Involving Human and/or Animal Rights Not applicable.
Informed Consent Not applicable.
Ethical Approval Not applicable.
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