## 4 Results and Discussion
### Antibacterial Test  
The antibacterial test of TM-PEC films and TM-nanoemulsion against _E. coli_ and _S.aureus_ were tested by using the viable cell-counting method. The antibacterial activities of thyme essential oil against _E.coli_ and _S.aureus_ have been investigated previously [65; 66; 67]. Figure 7 shows quantitative amounts of survival _E. coli_ for PEC films (with and without drug) and TM-nanoemulsion. The number of bacteria in TM-nanoemulsion decreased in comparison with that without thyme oil. This decrease was significant for TM-nanoemulsion of 2 v/v% indicating the dependency of antibacterial activity on concentration of nanoemulsion, which can be related to the hydrophobicity of the thyme oil due to the hydrophobic phenolic constituents such as carvacrol and thymol. The hydrophobic thyme oil diffuses in the lipid layer of the cell membrane, which could lead to deterioration and increase permeability of the membrane [68]. It has been assumed that the phenolic components, such as thymol and carvacrol, suppress the calcium and potassium transport by partitioning in the lipid phase of the membrane and subsequently changing the local environment of calcium channels [69]. Calcium has an established role in the normal homeostasis of mammalian skin and serves as a modulator in keratinocyte proliferation and differentiation [70].  
Figure 6: Cell viability of the PEC films: (1) TM-nanoemulsion, (2) PEC 1, (3) PEC 0.4, (4) TM-PEC %0.4, (5) TM-PEC %1.  
The number of _E.coli_ in TM-PEC films also decreased in comparison with that of negative control but was comparable with the initial number of bacteria (inoculum) indicating that the TM-PEC films could limit the growth of bacteria (\(p<0.05\)). Adding more chitosan did not have an obvious effect, which may be explained by the interaction between positively charged chitosan molecules and negatively charged microbial cell membranes. It has been shown that higher positive charge leads to the stronger antibacterial activity. The formation of polyelectrolyte complexes is mainly because of an electrostatic mechanism, so positive charge density on chitosan decreased. The electrostatic interactions between the polycationic structure and the anionic components of the microorganisms' surfaces is a basic factor in the antibacterial activity of chitosan [42].  
The quantitative amounts of survival _S.aureus_ bacteria for PEC films (with and without drug) and TM-nanoemulsion are also shown in Figure 8. Same results were observed. The number of bacteria in TM-nanoemulsion decreased in comparison with that of without thyme oil. This reduction was significant for TM-nanoemulsion of 2 v/v% indicating the dependency of antibacterial activity on concentration of nanoemulsion (same as _E.coli_). The number of _S.aureus_ in TM-PEC films also decreased in comparison with that of negative control but was comparable with the initial number of bacteria (inoculum) showing that the PEC films could limit the growth of bacteria. In all cases, a solution without any PEC film (containing only _E.coli_ or _S.aureus_ bacteria solution) was chosen as the negative control.  
It should be mentioned that in the antibacterial tests, although the growth medium is not similar to the wound exudates, it simulates the worst condition where the number of bacteria is much higher than may present at a wound surface. The prepared growth culture provides the best conditions in  
Figure 8: Antibacterial activities of TM-PEC films and TM-nanoemulsions against _S.aureus_.  
Figure 7: Antibacterial activities of TM-PEC films and TM-nanoemulsions against _E.coli_.  
terms of medium composition, pH, and temperature for the growth of test microorganisms, which leads to the maximum growth and activity of the microorganisms. Therefore, it can be concluded that higher activity is expected in real cases as wound surface and exudates do not maintain optimum growth conditions for the microorganisms. On the other hand, the natural defense mechanism during healing process also helps to prevent the growth of bacteria [71].