Graphene Decorated Zinc Oxide and Curcumin to Disinfect the Methicillin-Resistant Staphylococcus aureus

Sometimes, life-threatening infections are initiated by the biofilm formation facilitated at the infection site by the drug-resistant bacteria Staphylococcus aureus. The aggregation of the same type of bacteria leads to biofilm formation on the delicate tissue, dental plaque, and skin. In the present investigation, a Graphene (Gr)-based nano-formulation containing Curcumin (C.C.M.) and Zinc oxide nanoparticles (ZnO-NPs) showed a wide range of anti-microbial activity against Methicillin-resistant Staphylococcus aureus (MRSA) biofilm and demonstrated the anti-microbial mechanism of action. The anti-microbial effect of GrZnO nanocomposites, i.e., GrZnO-NCs, suggests that the integrated graphene-based nanocomposites effectively suppressed both sensitive as well as MRSA ATCC 43300 and BAA-1708 isolates. The S. aureus inhibitory effect of GrZnO-NCs improved >5-fold when combined with C.C.M., and demonstrated a M.I.C. of 31.25 µg/mL contrasting with the GrZnO-NCs or C.C.M. alone having M.I.C. value of 125 µg/mL each. The combination treatment of GrZnO-NCs or C.C.M. inhibited the M.R.S.A. topical dermatitis infection in a mice model with a significant decrease in the CFU count to ~64%. Interestingly, the combination of C.C.M. and GrZnO-NCs damaged the bacterial cell wall structure, resulting in cytoplasm spillage, thereby diminishing their metabolism. Thus, owing to the ease of synthesis and highly efficient anti-microbial properties, the present graphene-based curcumin nano-formulations can cater to a new treatment methodology against M.R.S.A.

and stored at −80°C till use. 0.4mM of Menadione solution was primed and filtered immediately just before the commencement of assay. Adherent bacterial cells were washed with PBS (200 μL), followed by addition of XTT, and two μl of menadione to each well. The solution was transferred to a new plate after incubation in the dark for four hrs at 37°C, and the colorimetric change in the solution was assessed using a microtitre plate reader (BIORAD Microplate reader at 490 nm) 1 . Experiments were performed in triplicate. The data were expressed as mean ± SD.

MRSA viability assay employing SYTO9 and propidium iodide (PI) dyes
To study the cell viability upon treatment with GrZnO-NCs and CCM combination the SYTO9-PI bacterial viability kit (Invitrogen, CA) was used. An aliquot (10μL) comprising of an equal proportion of SYTO9 and PI mixture was added to the treated cells in the six well-plates and incubated for 15 min in the dark at room temperature. After the stipulated incubation time, the cells were washed with cold, sterile PBS and mounted on a glass slide. The cells were seen under a fluorescence microscope(Zeiss, USA) 2,3 .

Invivo animal infection
Six to eight-week-old male BALB/c mice were used in the study. Five different groups were organized, having six animals in each group. Firstly, animals from each group were anesthetized by intraperitoneal (IP) injection of a ketamine-xylazine cocktail and subsequently shaved on the dorsal surface. The skin of the mouse was rasped with sterile scalpel blades until a reddened area appeared.
The surface of each wound was inoculated with 50 μl of the culture suspension corresponding to 10 8 CFU of MRSA ATCC BAA 1708 strain. The infection was allowed to establish for 96 h. The treatment schedule was followed as: 1. Group 1: Healthy mice exposed to normal saline only.
2. Group 2: Mice exposed to MRSA ATCC BAA 1708, followed by treatment with normal saline only.
5. Mice exposed to MRSA ATCC BAA 1708 followed by treatment with GrZnO-CCM (0.5 g/kg body weight). For histopathological examination, the skin tissues were taken out from the mice belonging to the different treatment groups followed by fixing in 10% formaldehyde, got dried out in variable concentrations of ethyl alcohol then, cleared in xylol and mounted in molten paraplast at 58-62 °C.
The finely cut thin segments were stained with hematoxylin and eosin dyes and assessed for any morphological changes under an Olympus BX40 magnifying lens (PA, USA) for the relative investigation 3,5 .

Ethical conduct of research
In this investigation, ingrained BALB/c mice (6-2 months old, 20 ± 2 g) were supplied from the Institute's Animal House Facility. All mice were housed in polypropylene confines and kept up ideal temperature conditions on a 12hr light-dark cycle and had free access to food and water. Every animal-based trial was performed by the rules of the National Regulatory Committee for Control and Supervision of Experiments on Animals (CPCSEA), and all the studies were specifically approved by the ethics committee. Our research institute endorsement ID is 332/CPCSEA for the department of Biochemistry (JNMC unit).