Antibacterial Activity of New Compounds Based on Nanocomposites

Background: Antimicrobial resistance is a major problem in treatment and public health, and it has been increasing over the last few decades. Hence, serious measurements are needed to overcome this challenge. In this study, we evaluated antibacterial and antifungal activity of some nanocomposites including titanium dioxide (5a), polyimide nanocomposites containing cerium oxide (5b), silver-titanium dioxide nanoparticles prepared under desired conditions (5c), polyaniline/wheat husk ash PANI /WHA (5d), Ag-TiO2 prepared by sol–gel route (5e), and cellulose-graphene (5f) against some bacterial and fungal strains, which are the most common agents in many infectious diseases. Methods: The nanoparticles were prepared in desired condition. The agar dilution and well agar diffusion methods were used for determination of inhibition zoon and minimum inhibitory concentration (MIC) during preliminary evaluation of antimicrobial activity against Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. Results: The results showed that the nanocomposites had good activity against gram-negative bacteria. Conclusions: Our results showed that the inhibitory activity of these nanocomposites on gram-negative bacteria was better than gram-positive bacteria.

dianhydride (PMDA, 0.1524 g) were stirred for 2 hours at 80°C and 400-500 rpm. After 2 hours of preparing both the suspension and polyamic acid, the suspension was added to polyamic acid, then stirred for 24-48 h to get the condensed material at the same rate and temperature. In this stage, the magnet could not move within the material. The material was discharged into a crucible and the fourstage cooking was performed as follows: 1 h at 100°C, 2 hours at 200°C, 1 hours at 250°C, and 1 hour at 300°C (14). To obtain Ag-TiO 2 nanosol, photodeposition of silver on TiO 2 NPs was executed by adding a desired volume of aqueous AgNO 3 (10 -3 M) followed by irradiation for 120 minutes with visible light source (200 W halogen lamp). Finally, the product was centrifuged and washed with distilled hot water and organic solvents to remove the adsorbed impurities, and then calcined for 3 hours at different temperatures (15,16). Polyaniline/wheat husk ash nanocomposite was prepared by adding 1 g of KIO 3 to 100 mL of sulfuric acid (1 M) and stirred by magnetic mixer until a uniform solution was obtained. Then wheat husk ash (1 g) and fresh distilled aniline monomer (1 mL) were added to stirred solution one after the other at room temperature for 5 hours.
Consequently, to separate the oligomers and impurities, the filtered product was washed several times with deionized water and then dried in oven for 24 h at 60°C (17). Inside the beaker, 4.5 g of cellulose and 0.5 g of graphene and acetone as a solvent were poured, and the solution was placed on heater while stirring until dry and then cellulose-graphene nanocomposites were obtained (18). Table 1 shows the size of NPs.

Antimicrobial Screening Microbial Cultures and Growth Condition
Standards collection of Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Streptococcus pyogenes, and Candida albicans were obtained from Urmia University of Medical Sciences, Iran. All the test strains were retained on nutrient agar slants (Sigma Aldrich, the US) at 37 o C and subcultured on nutrient broth (Sigma Aldrich, the US) for 24 hours prior to antibiogram test. These bacteria and fungi were used as important pathogens in estimating the antimicrobial nature of the synthesized NPs (19).

Agar Diffusion Method-Individual Compounds Solution
The antibacterial activity of nanocomposites was determined according to Clinical & Laboratory Standards Institute (CLSI) (20). The prepared NPs were dissolved in 100% dimethyl sulfoxide (DMSO) to final concentrations of 5, 2.5, and 1 mg/mL. Bacterial suspension with the turbidity of 0.5 McFarland was spread on Mueller-Hinton agar (MHA) plates with a sterile swab (19). Afterward, wells of 8 mm diameter were punched into the agar medium using a sterile borer and were loaded and filled with 70 μL of nanocomposites and allowed to diffuse at 37 o C for 24 hours and at 25 o C for 48 hours for bacterial and fungal loads, respectively. After incubation, the diameters of the growth inhibition zones were measured in mm. All experiments were performed with three replications for all mentioned microorganisms. Data were expressed as mean ± standard deviation (21,22).

Agar Dilution Method
Our plates containing defined concentrations of nanocomposites were prepared by using a single stock of each nanocomposite and pouring the molten agar into petri dishes. Experiments were performed with six different concentrations of each NP (1000 µg/mL, 500 µg/ mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL, and 31.25 µg/mL) to prepare doubling serial dilutions of nanocomposites in medium up to the sixth concentration. Negative control plates were prepared at the same way (23). Suspension of each microorganism with turbidity of 0.5 McFarland was transferred on MHA containing different concentrations of the nanocomposite to see their antimicrobial power. The growth of microorganism on MHA indicated that specific concentrations of NPs were unable to prevent the growth of the desired bacteria or fungi. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of an antimicrobial that inhibited the visible growth of a microorganism after overnight incubation. Also, minimum bactericidal concentration (MBC) was identified by determining the lowest concentration of antimicrobial agent that reduces the visibility of the initial bacteria or fungal inoculum by a preset reduction such as ≥99.9% (24). To get MBC and minimum fungicidal concentration (MFC), a loopful was taken from the MIC wells and streaked on plates. The growth was observed after incubation at 37 o C for 24 hours and at 25 o C for 48 h for bacterial and fungal loads, respectively (25).
In present study the increasing of concentration of nanocomposites from 1 to 5 mg/ml lead to higher antibacterial activity against Escherichia coli , Pseudomonas aeruginosa an Streptococcus pyogenes from 12-15 mm. For bacteria, the MICs of the compounds 5a, 5b, 5c, and 5d ranged from 400 to 800 µg/mL, while the MIC of the compounds 5e and 5f containing Ag-TiO 2 and cellulose/ grapheme were determined in the range of 200 to 800 µg/ mL. The MICs and MBCs values were similar for bacteria growth but about Candida. albicans, it was inhibited only by Ag-TiO 2 and minimal fungicidal concentration and MIC values were the same.

Discussion
In this study, the antimicrobial activity of some nanocomposites including TiO 2 (5a), polyimide nanocomposites containing cerium oxide (5b), silvertitanium dioxide NPs prepared under desired conditions (5c), polyaniline/wheat husk ash PANI /WHA (5d), Ag-TiO2 prepared by sol-gel route (5e), and cellulose-graphene (5f) were evaluated against important human pathogens. According to the results, gram-negative bacteria appeared to be more tolerant to silver than gram-positive bacteria (Table 1), except for S. pyogenes. Among six synthesized nanocomposites in this research, only Ag-TiO 2 prepared by sol-gel route (5e) had an inhibitory effect on C. albicans. According to the results shown in Table 1 and 2 , the order of inhibitory power was found to be 5e>5f>5c>5a>5d. Ag-TiO 2 nanocomposite prepared by sol-gel 5e route exhibited stronger bactericidal effects than the silvertitanium dioxide prepared under desired conditions 5c. Overall, recent findings show that the most important reason is the difference in particle size (26). The size of NPs is so critical for antimicrobial efficacy, so that as the size of NP becomes smaller, its efficiency in inhibiting the growth of microorganisms increases (27). The results of our report showed that antibacterial efficiency of Ag-TiO 2 was better than TiO 2 and these differences have also been found in recent studies (28). The Ag-TiO 2 NPs prepared by sol-gel route (5e) had higher efficiency in iinhibition of the growth of various microorganisms. Interestingly, the antibacterial activity of cellulose-graphene (5f) against most bacteria was equal to Ag-TiO 2 (5e). Although a comparative study of MIC values indicated that the inhibitory activity of these nanocomposites on gram-negative bacteria was better than gram-positive bacteria, the study conducted by Xie et al showed that the cellulose/graphene coated oxide-CuO nanocomposite films displayed significant antimicrobial activities against gram-positive bacteria (29). Haghi et al demonstrated the inhibitory influence of TiO 2 nanoparticles on the growth of pathogenic strain of E. coli and and proposed the manufacture use of titanium dioxide on various surfaces in hospitals to prevent infections (30). The results of an interesting comparative study between the antibacterial nature of TiO 2 and Ag-TiO 2 , CuO-TiO 2 showed that Ag and Cu alone have maximum inhibitory activities, but combinations of Ag or Cu with TiO 2 were more active than TiO 2 (31). The difference in the structure of the cell membrane was one of the most important factors in this regard. In fact, the thick layer of peptidoglycan in gram-positive bacteria acts as a strong protector against external factors and protects the cytoplasmic membrane, but gram-negative bacteria do not have this layer (28,32). The NP diffusion rates was another important factor affecting antimicrobial activity (33).

Conclusions
Due to the antibacterial activity of 5e and 5f nanocomposites against Escherichia and Pseudomonas, they can be used as important antimicrobial agents in textiles and biomedical devices for continuously releasing NPs to provide protection against infectious agents in hospital equipment.

Conflict of Interests
None.