Interaction of bacteria with graphene oxide particles reduces their ability to biofilm formation on PVC Microplates

. Progress in the diagnosis and treatment of human diseases is impossible without the use of catheters and implants in contact with the skin, mucosal epithelium and blood of the patient. An important task in the manufacture of implantable medical devices is their resistance to protein biofouling and the formation of bacterial biofilm on their surface. The interaction of bacteria with graphene oxide particles contributed to a decrease in the number of Staphylococcus aureus (48.6±1.7% CFU) and Escherichia coli (29.7±2.1% CFU) bacteria. Incubation of Staphylococcus aureus and Escherichia coli cells with graphene oxide particles resulted in a decrease in the ability to biofilm formation on 96-Well Clear PVC Microplates. The creation of composite materials based on polyvinyl chloride and graphene may be one of the strategies for reducing biofilm formation on the surface of implantable medical devices.


Introduction
Today, the use of implantable medical devices is a common medical procedure used for diagnostic and therapeutic purposes.
PVC (polyvinyl chloride) has proven itself as a reliable, safe and economical material for 60 years of use in clinical practice. The properties of polyvinyl chloride -simple production technology, transparency, chemical stability when interacting with biological fluidscontribute to the fact that pre-sterilized disposable medical products are made from PVC. The range of applications for PVC in clinical practice is wide, from soft blood bags, urine continences, containers for intravenous solutions, oxygen masks, endotracheal tubes, to examination and surgical gloves. Catheters and cannulas made of PVC meet all the requirements necessary for clinical use -flexibility, transparency, strength, kink resistance and suitability for sterilization.
Despite the fact that tubes made of other materials (latex, silicone ...) are used in clinical practice, polyvinyl chloride tubes are the "gold standard". In fact, the only but important problem when using PVC medical devices in clinical practice is the short time of their usedue to the adhesion of plasma proteins, immunocompetent cells and bacteria. Therefore, one of the primary tasks facing scientists is to increase the time of using PVC medical products -the creation of coatings or composite materials with anti-adhesive and bactericidal properties that prevent the adhesion of serum proteins, eukaryotic cells and bacteria.
Today, this task is complicated by the increasing resistance of bacteria to most antibacterial drugs and antiseptic substances used in clinical practice. Therefore, the search for strategies to reduce bacterial contamination and biofilm formation on the surface of catheters and implantable devices is one of priorities [1].
Thus, one of strategies to prevent the formation of bacterial biofilm on medical devices is to modify their surface. Produced anti-biofilm materials can be divided into engineered surfaces with nano-and micro-relief that inhibit bacterial adhesion [2], materials with surfaces leached by a biocide [3] and materials with immobilized antibiotics [4]. However, the use of materials with immobilized antibiotics leads to the emergence of multidrugresistant bacteria that cause nosocomial infections.
In addition, doctors in dental practice often encounter infectious complications (stomatitis) when wearing dentures for a long time. Thus, it was noted that wearing polymer bridges for more than 6 months not only contributes to the restoration of physiological functions (protective, chewing, aesthetic, communicative), but can be a source of infectious complications leading to failure of orthopedic treatment.
Graphene, more precisely (GBNs -graphene-based nanomaterials) are "new materials" that are characterized by a two-dimensional planar network structure, rich in oxygencontaining functional groups, easy surface modification, good hydrophilicity and biocompatibility. They have excellent electrical, photothermal and mechanical properties. Biosensors (made from graphene-based nanomaterials) are used to detect RNA/DNA, peptides, hormones, glucose, adenosine triphosphate (ATP), and hydrogen peroxide [5]. Graphene-based nanomaterials are widely used in molecular sensors, new energy sources, transistors, chemical catalysis, solar cells, and electrochemistry.
Today, all over the world, there is an intensive development of materials preventing bacterial colonization of the surface, materials with low bacterial adhesion.
Despite the fact that many methods are described in the scientific literature for evaluating the effectiveness of such surfaces [6,7]. However, due to the lack of standardization of these methods, the results obtained by different researchers are difficult to compare with each other. One of the solutions to this problem can be an interdisciplinary approach based on the fact that the physicochemical properties of the material, biocompatibility and antibacterial properties are evaluated in the same methodological way.
Our study is devoted to the analysis of the effect of graphene oxide particles on the ability to biofilm formation by clinical strains of Staphylococcus aureus and Escherichia coli on PVC Microplates plates.

Bacteria
We used biofilm-forming strains of clinical isolates of bacteria from the urogenital tract in urinary tract infections (Escherichia coli (10 strains) and wound discharge -Staphylococcus aureus (10 strains). Bacterial suspensions were prepared in 0.15 M NaCl solution and standardized according to the McFarland standard test (0.5 units). Subsequently, bacterial suspensions were diluted 1:100 and working solutions were made on their basis.

Biofilm formation
Biofilm formation was studied using the photometric method. For clinical isolates of Staphylococcus aureus, the Knobloch J.K. et al. [8], and for Escherichia coli cells, the O'Toole G.F. et al. [9]. The intensity of dye extinction corresponded to the degree of biofilm formation. The optical density was measured on a KFK 2 MP concentration photoelectric colorimeter (AO «ZOMZ», Russia), at λ=540 nm.

Interaction of bacteria and particles of graphene oxide
To study the resistance/sensitivity of bacteria to graphene oxide particles, we used dilutions of graphene oxide powder in 0.15 M NaCl solution at a final concentration of 0.5 μg/mL. Bacteria and graphene oxide (GO) particles were incubated in a thermostat (37°C, 60 minutes). After incubation, the experimental solution (50 μl) was applied to the surface of a solid nutrient medium. After that, the bacteria were grown in a thermostat (37°C, 24 hours). The calculation of the number of CFU on solid nutrient medium was carried out on the next day.

Statistical methods
The results obtained were subjected to statistical processing by methods of variation statistics.

Results and discussion
Graphite oxide (GO) is a compound with a layered structure, obtained by the oxidation of natural graphite with a high degree of crystallinity, and is easily dispersed in H2O. Each graphite particle has a dense two-dimensional carbonaceous skeleton, which is built from a large number of sp 3 -(C) and a smaller number of sp 2 -(C). In a colloidal dispersion, each particle has a size of 5 to 10 nm. With a change in the composition of the colloid, with the addition of salt, as we used in our work (0.15 M NaCl solution), the state of the particles changes. There are at least two types of secondary conformations of graphite particles in which they are found in solutions containing salts. Formation in a solution of a large-scale particle "laminated layer aggregate", consisting of many thin-film particles stacked on top of each other. Each straight section of the "laminated layer of the aggregate" contains a large bend, with each primary particle also sharply curved.
Another type of secondary conformation of graphene particles is called "randomly shaped aggregate" (Fig. 2). The image shows that the aggregates of thin-film particles are unevenly bent and deformed, like crumpled paper. This indicates that the affinity between the particles and the dispersion medium is very low and the particles aggregate like a linear flexible polymer. This may reduce the degree of their interparticle orientation and may lead to particle aggregation. These features of the behavior of graphene particles in solutions containing salts must be taken into account due to the possible aggregation of particles and a decrease in the adhesion of bacteria on the surface of graphene oxide particles. At the first stage of our study, we tried to evaluate the bactericidal effect of graphene oxide particles upon incubation of graphene oxide (GO) particles with gram-negative (Escherichia coli) and gram-positive (Staphylococcus aureus) bacteria. It was impossible to assess the bactericidal effect of graphene oxide particles in solution by optical density, since graphene oxide (GO) colored the solution containing a suspension of bacteria and graphene oxide powder black. Therefore, after incubation (37°C, 60 min) of the experimental mixture (0.5 ml (bacterial suspension) + 0.5 ml (graphene oxide solution 0.5 μg/ml), aliquots (50 μl) were taken and rubbed with a spatula over the surface of a solid nutrient medium. In the control sample, instead of graphene oxide was used in 0.15 M NaCl solution. On the next day, the number of CFU was counted and the index of bacterial sensitivity was calculated by the formula: Index (sensitivity) = 100 -CFU (experiment) / CFU (control) × 100 (1) As a result of calculating the sensitivity index (1), we noted that the sensitivity index of Staphylococcus aureus cells to graphene oxide particles was characterized by inter strain variability -from 43% to 59% CFU (Fig. 3), but on average it was high (48.6±1.7 % CFU).
For strains of Escherichia coli with high inter strain variability -from 17% to 39% CFU, the sensitivity index was significantly lower -29.7±2.1 CFU.   At the second stage of the study, we were tasked to investigate the effect of graphene oxide (GO) on the ability of bacteria to biofilm formation in the wells of polyvinyl chloride plates. We used the same clinical bacterial isolates (10 strains of Escherichia coli and 10 strains of Staphylococcus aureus) in our work.
For clinical strains of Staphylococcus aureus, the thickness of biofilm estimated by dye extinction varied significantly (from 0.22 OD to 0.41 OD). In addition, 3 out of 10 strains of Staphylococcus aureus did not form biofilm. Mean values of biofilm formation by Staphylococcus aureus cells were 0.32±0.03 OD (Fig. 4A).
The ability to form biofilms in the wells of polyvinyl chloride plates for clinical strains of Escherichia coli varied significantly (from 0.19 OD to 0.39 OD). The mean values were 0.29±0.02 OD (by dye extinction). Bacteria were incubated with graphene oxide particles as described by us earlier. The ability to biofilm formation after interaction with graphene oxide particles decreased both for Staphylococcus aureus (0.24±0.03 OD) and Escherichia coli (0.23±0.02 OD) cells (Fig. 4B).

Conclusions
Thus, the interaction of bacteria with graphene oxide particles contributed to a decrease in the number of Staphylococcus aureus cells (by 48.6±1.7% CFU) and Escherichia coli (by 29.7±2.1% CFU). Incubation of Staphylococcus aureus and Escherichia coli cells with graphene oxide particles resulted in a decrease in the ability to biofilm formation on polyvinyl chloride plates. The methodology and results of our study can be used to assess the adhesion of bacteria and biofilm formation of when testing new composite materials based on polyvinyl chloride and graphene. Some of these composite materials are already under investigation. Thus, a technique has been developed for obtaining a composite material based on graphene-polyvinyl chloride and graphene-organosilane. Graphene oxide particles have become part of polymer and chitosan composites that can be used for biomedical applications. One of the promising applications of graphene can be the production of composite materials using additive technologies.
Thus, coating with a graphene layer of hydrophobic materials (Poly(dimethylsiloxane) (PDMS), MakerBot polylactic acid (PLA), MakerBot acrylonitrile butadiene styrene (ABS), Stratasys Object acrylates and acrylics) produced by three-dimensional (3D) printing methods and used for the manufacture of implants can significantly reduce protein fouling and adhesion of immunocompetent cells and bacteria. In the future, this may become one of the strategies for chemical modification of the surface of implantable medical devices.
In dental practice, prosthetic stomatitis is one of the most common oral diseases in the elderly. This disease is associated with prolonged wearing of crowns and bridges, often contaminated with periodontopathogenic bacteria (Prevotella intermedia, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium periodonticum, Actinomyces naeslundii, Streptococcus intermedius) and Candida albicans. The adhesion of bacteria of the periodontopathogenic group was significantly reduced on "tablets" prepared from NextDent C & B Micro Filled Hybrid and Detax Freeprint temp UV photopolymer resins in comparison with self-polymerizing composites Luxatemp Automix Plus and Acrytemp polymers [11]. The addition of graphene oxide particles to photopolymer resins will significantly reduce the number of adherent bacteria due to the bactericidal effect.
Today, there are no unified methods for evaluating materials used for the manufacture of medical catheters and implantable devices. This does not allow comparing the results obtained using different methods and does not allow one to judge the commercial prospects of new materials for medical implantable devices, including those based on graphene.