In vitro assessment of activity of graphene silver composite sheets against multidrug-resistant bacteria and Tomato Bushy Stunt Virus

Purpose: To synthesize graphene-based silver nanocomposites and evaluate their antimicrobial and anti-Tomato Bushy Stunt Virus (TBSV) activities. Methods: A graphene-based silver composite was prepared by adsorbing silver nanoparticles AgNPs to the surfaces of graphene oxide (GO) sheets. Scanning electron microscopy was used to analyze the morphology of the synthesized graphene-based silver nanocomposite. This compound was investigated for its antimicrobial activity against several multidrug-resistant human pathogens using the agar well diffusion technique. Moreover, the biocompatibility and antiviral activity of the graphene-based nanocomposite against TBSV was studied in lettuce. Results: The graphene-based silver composite exhibited remarkable antimicrobial effects against pathogenic bacteria including Shigella sonnei and Pseudomonas aeruginosa with zones of inhibition of 32 ± 0.11 and 29 ± 0.05 mm, respectively. They inhibited TBSV better than graphene and GO. Conclusion: The synthesized graphene-based silver composite exhibits potent activity against TBSV and multidrug resistant bacteria, indicating that they are good candidates for future therapeutic applications.


INTRODUCTION
The most versatile application for graphene is its use in composite materials.Low production costs make graphene-based composites attractive and competitive for a variety of uses [1][2][3].The most important characteristics of graphene oxide (GO) are low cytotoxicity, good colloidal behavior, large surface area and low cost [3].GO is applied in medicine, agriculture, food, aerospace, electronics and defense industries [4].The literature reports positive effects of carbon nanotubes on plant growth and development, such as increased of root growth of onion cucumber and rye-grass in response to carbon nanotubes [5].The nanotubes likely stimulate cell division and plant growth by activating aquaporins and major gene regulators of cell division and extension [6].
Tomato bushy stunt virus (TBSV) is distributed all over the world, including Central and Western Europe, North Africa and the USA [7].Dieback and necrosis of romaine and leaf lettuce caused by TBSV TBSV-have become increasingly important in California and the incidence is becoming more widespread [8,9].Recently, nanoscale materials have been raised as novel microbial killers because of their unique chemical and physical properties and their large surface area to volume ratio.The antimicrobial characteristics of graphene-based composite materials have promoted their use in medical and ecological applications and various consumer goods [10].
The present work investigated the development and analysis of graphene-based antimicrobial and antiviral agents.

EXPERIMENTAL Preparation of graphene oxide
Graphite (5 g), 2.5 g NaNO 3 and 110 mL H 2 SO 4 were mixed at 0 °C.Thereafter, 15 g of KMnO 4 were added.The suspension was diluted with 500 ml of 25 °C water and treated with 100 -200 mL 30 % H 2 O 2 to reduce the residual KMnO 4 .When the suspension turned bright yellow, it was filtered and washed.The resulting yellowishbrown cake was vacuum dried overnight at room temperature [11,12].

In-situ preparation of nanosilver on graphene oxide
GO (1.0 g) was dispersed in 75 ml double distilled water.To this solution, the required amount of silver nitrate and tri-sodium citrate were added {(AgNO 3 ) : (trisodium citrate) = 2}.The solution was heated at 130 °C with vigorous stirring for 60 min.The highly-viscous solution was cooled to −5 °C.The excess water was removed under vacuum and the product stored in a dry place.

Test microorganisms
The antimicrobial activity of graphene, GO

Agar-well diffusion method
The antimicrobial potential of graphene, GO and GO-AgNPs against the selected isolates was evaluated using agar-well diffusion assay.Fresh overnight cultures of the bacteria (100 µL) were inoculated into Muller-Hinton broth and incubated at 30 °C in a shaking incubator.After incubation, the optical density was adjusted to 0.5 McFarland standards, then 1 mL (10 6 CFU/mL) was swabbed onto Mueller Hinton II-agar plates and allowed to dry for 5 min.Afterwards, graphene and graphene-based molecules were placed in a well in the agar using a sterile micropipette.Plates were incubated overnight at 37 °C and the inhibition zone diameter (IZD) were measured using Clinical & Laboratory Standards Institute guidelines.

TBSV
was collected from random Capsicum annuum (pepper) samples from different fields in Egypt in 2016.Samples were tested for TBSV by the tissue blotting technique (TBPA) as described by [13,14], using polyclonal antibodies specific for TBSV (Agdia, Elkhart, IN, USA).Trans-Blot nitrocellulose membranes (BioRad Laboratories, CA) were used in this study.

Propagation of Egyptian isolate of TBSV
Infected sap was prepared from upper infected leaves and used for inoculation in 0.01 M sodium phosphate buffer (pH 7.0).The virus was biologically purified using the single local lesions system and further propagated in lettuce (Lactuca sativa) by mechanical inoculation.

Greenhouse experiments
Susceptible cultivars from lettuce seeds were germinated and grown in pots.After 25 days of planting, Lettuce seedlings were inoculated with TBSV.Four solutions were prepared containing buffer (control), graphene (Gr 1 mg/mL), GO-AgNPs (1 mg/mL) and GO (12, 6, 3 and 1.5 µg/mL), respectively Lettuce plants were inoculated in a randomized block design with three seedlings per pot, one pot per replicate, and four replicates per treatment (total 12 seedlings per treatment).The TBPA test and observation of symptoms were used for checking the presence of the virus.

Statistical analysis
Data were analyzed using SPSS v. 22 software, and the results are presented as mean  SD (n = 3).Comparison of means between different groups was performed using one-way analysis of variance.Statistical significance was defined as p  0.05.

Characteristics of Gr, GO and GO-AgNPs
Fig. 1A shows the regular graphene oxide sheet.Fig. 1B shows scanning electron microscope images of the synthesized hybrid; AgNPs were deposited uniformly on the GO surface with size distribution 30 -50 nm.The resultant hybrids formed well-dispersed aqueous colloids.

Antimicrobial activity
Table 1 shows the results of antimicrobial assay of Gr, GO and the GO-AgNP composite.After incubation at 37 °C for 24 h with Gr or GO, no inhibition zone was observed for the fungus C. albicans.For GO-AgNP, an inhibition zone was obvious after incubation for 8 h.The GO-AgNPs were effective antimicrobials against all the bacteria tested, with similar activity against each tested strain.The antimicrobial activity of GO was much less than that of GO-AgNPs for all the strains.GO showed greater antibacterial activity than graphene.

Source of virus inoculum
Naturally infected pepper leaves showing symptoms of TBSV infection (dieback, necrosis and bushy stunting) were collected from fields in Egypt (Figure 2) and gave a positive reaction in the TBPA test for the presence of TBSV using TBSV-specific polyclonal antibodies.

Propagation of Egyptian isolate of TBSV
Pepper leaves with typical viral symptoms were used to prepare sap for inoculation.The virus was transferred into the local lesion host Gomphrena globosa.Single local lesions were isolated and propagated further in lettuce (L.sativa) by mechanical inoculation.Infected lettuce plants which give positive reactions in the TBPA test became sources of TBSV for testing the activity of Gr and derivatives against TBSV.

Antiviral activity of Gr, GO and its AgNP composite
In vivo screening of the antiviral activities of Gr, GO and its AgNP composite against TBSV was performed using a local lettuce cultivar.Typical symptoms of TBSV were observed in inoculated lettuce seedlings compared with non-inoculated plants.Severe viral infection was observed for the control infected treatment.Spread treatment with graphene and the GO-AgNP composite reduced the TBSV symptoms in inoculated lettuce seedlings.These treatments also decreased the virus concentration, infection percentage and disease severity of inoculated lettuce seedlings in comparison with infected controls.Table 2 and Figure 3 show that postinoculation treatment with GO-AgNPs resulted in a higher activity against TBSV than was observed for GO.The data in Table 2 indicate that the effect on disease severity did vary depending on the GO concentration.

DISCUSSION
AgNPs were uniformly deposited on the GO surface with; the resultant hybrids formed welldispersed aqueous colloids.Similar findings were reported previously [16,17].
In antimicrobial tests, GO-AgNPs inhibited all the test bacteria, to approximately the same extent for each strain.The antimicrobial activity of GO was much less than that of the GO-AgNPs toward the same pathogens in the same conditions.Figure 3: Effect of graphene, graphene-based nanosilver and graphene oxide on a susceptible lettuce cultivar in the presence of TBSV in greenhouse conditions.T1: healthy control; T2 infected control; T3: graphene with TBSV; T4: graphene-based nanosilver with TBSV; T5: graphene oxide (12 µg/ml) with TBSV; T6: graphene oxide (6 µg/ml) with TBSV; T7: graphene oxide (3 µg/ml) with TBSV; T8: graphene oxide (1.5 µg/ml,) with TBSV These results correlate with the work reported by Gu et al [18], where addition of silver to graphene increased and prolonged its antimicrobial activity.Bare GO sheets are intrinsically bactericidal (bacterial survival percentage <1 % at 200 µg/ml) [19], resulting from the production of superoxide radicals, which caused oxidative stress, DNA fragmentation and loss of cell viability in P. aeruginosa [20].It has also been suggested that the antimicrobial properties of GO result both from oxidative stress and membrane contact [21].The enhanced antibacterial activity of GO-AgNPs may result from the high stability of AgNPs anchored on the GO sheets, and the positively charged hybrid surface, which increases electrostatic interactions of the bacterial cell membrane with the nanohybrid [22].Gram-positive bacteria are more sensitive to AgNPs than Gram-negative bacteria because of the interactions of positively charged AgNPs with negatively charged lipopolysaccharides on the bacterial surface [23].
Virus inhibition assays demonstrated that the activity clearly depends on the particle dimension and the spatial distribution of the interacting ligand/receptor molecules between coat proteins of the virus and infected cell receptors.GO and GO-AgNPs may enter the cell and exert antiviral activity through interactions with viral nucleic acids.
The results obtained in this study show that a GO-AgNP composite could be successfully prepared by decorating AgNPs onto the surfaces of GO sheets.The GO-AgNP composite possessed potent and desirable biological activities.The strongly-coupled interaction between Ag nanocrystals and GO, as well as the presence of the Ag dots, contributed to the antibacterial properties of the GO-AgNP composite.

CONCLUSION
GO -AgNPs composites have been prepared successfully by layering AgNPs on to the surfaces of GO sheets.The synthesized graphene-based silver composites represent a good lead for the development of potent antiviral agents against TBSV and multidrug resistance bacteria.

Table 2 :
Effect of different concentrations of graphene, graphene oxide and graphene-based nanosilver on virus appearance, Virus infectivity (I) and disease severity (DS) of a susceptible lettuce cultivar.