## 3 Results and Discussion
### Antimicrobial Activity  
Figure 9 shows the inhibitory halo achieved when the bacterial strains are exposed to antibiotics to confirm the effectiveness of the method. In contrast, no inhibitory effect was observed on the studied samples by using Kirby-Bauer method in the presence of _Escherichia coli_, _Enterococcus faecalis_ and _Staphylococcus aureus_. Figure 9: Inhibitory halo of antibiotics in contact with different strain ((**A**) _E. faecalis_, (**B**) _E. coli_ and (**C**) _S. aureus_) and chitosan/HNa discs in contact with: _E. faecalis_ (**D**), _E. coli_ (**E–I**) and _S. aureus_ (**J–L**). Many studies suggest that the antib biofilm property of chitosan is attributed to the polycationic nature of its N-H2 groups of the N-acetylglucosamine units. [20; 50; 51]. However, there are studies that indicate that the antibacterial activity of chitosan can be influenced by molecular weight, DDA and environmental conditions (e.g., the pH), type of derivative, solvent composition [50; 52], bacterial strain, culture media, temperature, ionic strength, metal ions, EDTA, and organic matter among others [51]. It is worth mentioning that all our films were at a neutral pH (\(7+0.5\)) and it is acknowledged that the antimicrobial and antibiofilm properties of chitosan and its derivatives are usually functional under the acidic condition [20], i.e., reducing its antimicrobial activity at pH 7.0 [51]. [PERSON] et al. indicate that Oleloyl-chitosan nanoparticle were antibacterial against _E. coli_ and _S. aureus_ by damaging the structures of the cell membrane and the alleged binding to extracellular targets such as phosphate groups or intracellular targets such as DNA. The hydrophilicity of the films, measured by contact angle, would provide a high affinity for water molecules, which would give our samples anti-fouling properties, as [PERSON] et al. pointed out [19]. However, in contrast to the results of [PERSON] et al., in our samples, no decrease in bacterial adhesion was observed. This could also be influenced by the topographic characteristics of the film surfaces, which are known to affect the adhesion process [19; 53; 54], being greater when the surface is rough [55; 56]. An increase in surface roughness stimulates the bacterial bond due to the increased contact area between the bacterial cells and the material's surface [54]. [PERSON] et al. [57] evaluated the antimicrobial activity of chitosan-LDPE films, against E. coli, _L. monocytogenes_ and _S. enteritidis_ by observing bacterial growth in the films with the lowest chitosan concentration. The antimicrobial action of chitosan may be due to its stacking on the surface of the bacterial cell wall, blocking the nutritional supply by changing the permeability of the cell wall. When the amount of chitosan released from the film matrix is insufficient, bacterial inhibition is limited or inexistent. [PERSON] et al. report that small concentrations of chitosan were unable to maintain lethal conditions against microorganisms and without complete inhibition, the recovery of microbial cells occurred over time [57]. Another explanation of our results obtained in the antimicrobial analysis may be due to the presence of sodium hyaluronate. [PERSON] and [PERSON] [58] suggested that the presence of sodium reduces chitosan's activity against _E. coli_. Sodium hyaluronate has an important effect on films and the low antibacterial activity may be due to a low density of CHT in the most superficial layer of the sample, leaving the HA exposed, promoting bacterial adhesion [59; 60]. [PERSON] et al. [51] conclude that the broth microdilution technique is the most appropriate to evaluate the potency of an antimicrobial agent by estimating the MIC (minimum inhibitory concentration), unlike the methods based on agar, so the bacterial growth should be well chosen, to avoid unfavorable interaction with chitosan. Figure 10 shows the antibacterial activity of PEGDE3 and GA crosslinked CHT/HNa 15% films against _S. triphimuriumium_.