*3.3. Effect of Exposure Duration on Formation of S. Aureus and E. Coli Biofilms*

Figure 8 shows OD600 values of eluted dye solution by *E. coli* and *S. aureus* for different duration of exposure (6 h, 9 h, 12 h, 15 h, 18 h, 20 h, 22 h and 24 h) of neat epoxy, epoxy/TiO2 (1 wt%) and epoxy/Ag-TiO2 (1 wt%). The biofilm ODs presented are averages of four independent experiments. Time course studies showed bactericidal ability of prepared composite surface up on contact and effectiveness in restraining bacterial biofilm formation. *S. aureus* biofilm formation response to time increased gradually, but it declined over a longer incubation period. It is plausible that this is due to biosorption of minerals and metals by microbial biofilms from the environment with which they are in contact [39,40]. When higher levels of silver is reached or with chronic exposure, it should be possible to limit the ability of the biofilm biosorption capacity, silver would then inhibit biofilm formation during prolonged exposure.

**Figure 8.** Growth curve for biofilm formation on neat resin, epoxy/TiO2 and epoxy/Ag-TiO2 composite of (**a**) *E. coli* and (**b**) *S. aureus*.

The results showed that biofilm formation was highly inhibited in a dose dependent manner as shown in Figure 9. Increasing the load of Ag-TiO2 resulted in shorter inhibition time *i.e*., antibiofilm activity of composite is directly proportional to Ag-TiO2 loading. Exposure of the composite with 1.5 wt% Ag-TiO2 for 24 h. resulted in a inhibition of 100% (as per crystal violet binding assay) of both *E. coli* and *S. aureus*. The higher activity of these composites against *E. coli* a Gram-negative bacterium is attributed to its thinner peptidoglycan cell wall compared to *S. aureus* a Gram-positive bacterium. Complete inhibition of biofilm was achieved with 24 h of irradiation time with composite of Ag-TiO2 with 1.5 wt% loading, in case of both *E. coli* and *S. aureus* (see Figure 9a,b)*.*The antibacterial activity could also have effect on planktonic bacteria due to silver that has diffused to media from the matrix. The bactericidal efficacy of these composite is through the diffusion of photogenerated ROS and Ag<sup>+</sup> particles (acting as a leaching biocide) to the surface from the bulk of the polymer where such species/particles attack proteins and membrane lipids in bacterial cell wall. The driving force for silver particle diffusion is determined by a concentration gradient, which forms between the bulk of the composite material and the surface. The diffusion behavior depends on several factors including the structure of the material, environmental osmolarity and temperature.

We have quantified the silver release characteristics at 37 °C for the composites loaded with the 0.5 wt% to 2.0 wt% Ag-TiO2 filler Table 1. And observed that non linear increase in the release of silver on increase of Ag-TiO2 loading. The total released silver from the coatings was 6.6 to 16.8 ȝg/mL (16.8 ppm) after 48 h by epoxy/Ag-TiO2 composites in the culture media without inoculum. From this observation it can be concluded that all the Ag-TiO2 containing composites can have antibacterial activity even in the dark due to release of silver. However, presence of UV light will hasten the bactericidal activity of the composite due to photogeneration of ROS. Similar observations were made by Akhavan and Ghaderi [41] who investigated bactericidal activity of the anatase-TiO2, the Ag thin film and the Ag-TiO2/anatase-TiO2 nanocomposite thin film against *E. coli* at dark and under UV exposure. In addition, they found superior antibacterial activity of Ag-TiO2/anatase-TiO2 nanocomposite thin film under the UV irradiation due its photocatalytic capability when compared to non-photocatalytic bare Ag and TiO2 films and the silver ions released by Ag-TiO2/anatase-TiO2 nanocomposite thin film became saturated after 20 days at ~2 nM/mL. It is also possible to regulate the release of silver to the desired concentration by varying the nano-filler load incorporated into polymer composites and by tuning Ag-TiO2 structure/composition during the sol-gel incorporation process. Antibiofilm activity of these composite remained unchanged at least for 5–6 cycles when we challenged during experiment through replications, this is due to continuous and uniform diffusion of the antimicrobial agents (ROS and silver species).

**Figure 9.** Biofilm inhibitory effect of Ag-TiO2 loading (dose response) after 6, 24 and 48 h of irradiation on (**a**) *E. coli* and (**b**) *S. aureus*.
