## 2 Co-Administration of Two Different Antimicrobial Polymers
### Improving Antimicrobial Activity of Chitosan by Grafting Biocidal Polymers  
Chitosan (CS) is a naturally occurring polymer obtained through the alkaline deacetylation of chitin possessing many attracting properties including antimicrobial activity and nontoxicity [23]. Further, CS has shown antimicrobial activity against a wide range of microorganisms, including bacteria, fungi and yeasts [23; 24; 25]. A widely accepted mechanism of biocidal action for this polymer involves the protonation of its amino groups at the acidic pH value (lower than pKa) that lead to the formation of cationic groups, which electrostatically interact with bacterial cell walls. Thus, at the acidic pH value, the antimicrobial activity of CS is attributed to the electrostatic interactions and chelation effects. Nevertheless, CS can also be modified to enhance its solubility and to improve its biocidal properties. For instance, [PERSON] et al. [26] investigated the antimicrobial activity of a dual system based on CS modified with guanidine derivatives. It is noted that guanidinium salts have also been investigated as medical and crop protection agents and antiseptics for industrial products due to their high antimicrobial and antifungal activity [27]. Research results highlighted that guanidinylated CS had a higher antibacterial activity, whose MIC in hydrochloric acid aqueous solution (pH 5.4) was four times lower than the one observed for pristine CS. Moreover, guanidinylated CS inhibited the growth of _S. aureus_ and _B. subtilis_ at pH 6.6. This enhanced capability of modified CS was attributed to an overall increase in the positive charge density as a consequence of the incorporation of guanidine derivatives, which promotes the adsorption of these polycations onto the negatively charged cells surface. Nevertheless, the quaternization of CS with alkylating agents is probably the most frequently employed approach to confer permanent positive charges and to improve biocidal activity [28]. Another common strategy is also the grafting of acidic derivatives, such as gallic and caffeic acids, onto CS via carbodiimide based coupling, free radical mediated grafting, among other synthetic approaches [29]. On the whole, most of the modified CS exhibits higher antimicrobial activity against Gram-positive and Gram-negative as compared to pristine CS [30; 31]. This could be ascribed to an increase in the permeability of these polymers through the outer membrane in the case of Gram-negative bacteria and the cell membrane in Gram-positive bacteria producing the release of the intracellular components [32]. Aside from the preparation of CS with various biocidal functions, CS has also been combined with other antimicrobial polymers via the grafting or blending processes. For instance, poly[2-(acryloyloxy)ethyl]trimethylammonium chloride, was grafted onto CS chains to improve its antimicrobial activity in coatings as tested against Gram-positive _Staphylococcus aureus_, Gram-negative bacteria _Klebsiella pneumoniae_ and _Aspergillus fumigatus_ fungi [33]. Recent works mentioned the functionalization of chitosan with \(N\)-halamines [34] and pyridinium compounds [35] grafted as side groups with respect to the chitosan main chain in order to enhance the biocidal activity, taking advantage of the antimicrobial features of each one. Moreover, low molar mass antimicrobial polymer, e.g., \(\varepsilon\)-poly-\(\iota\)-lysine (EPL), have also been grafted onto CS chains [36], to improve antimicrobial activity against Gram-negative bacteria (_Escherichia coli_ and _Pseudomonas aeruginosa_), Gram-positive bacteria (_Enterococcus faecalis_ and methicillin-resistant _S. aureus_ (MRSA)) and fungi (_Candida albicans_ and _Fusarium solani_). Although these latter grafted copolymers revealed lower activity than pristine EPL against bacteria, they exhibited superior antifungal activity. This effect suggests the occurrence of a synergistic effect. [PERSON] et al.