*3.5. Antimicrobial Properties*

Figure 13 reports the microbicidal effect vs. time profiles evaluated for CH, CH/CS and CH/HA scaffolds loaded with norfloxacin as a free drug (a, c) and (b, d) as nanocomposite at 1% against Pseudomonas aeruginosa and Staphylococcus aureus.

**Figure 13.** Microbicidal effect evaluated for 1% norfloxacin as a free drug (**a**,**c**) and (**b**,**d**) as nanocomposite loaded into CH, CH/CS and CH/HA scaffolds against Pseudomonas aeruginosa (**a** and **b**) and Staphylococcus aureus (**c** and **d**), in comparison to norfloxacin as a free drug and as nanocomposite, with the same concentration as in the scaffolds (mean values ± SD; n = 3).

*Pseudomonas aeruginosa* is a facultative Gram-negative anaerobe bacterium. It is recognized as a multidrug-resistant pathogen for its intrinsically advanced antibiotic resistance mechanisms since it causes infections of considerable medical importance, among them hospital-acquired infections such as sepsis syndromes. *Staphylococcus aureus* is facultative Gram-positive anaerobe bacterium. It is part of the skin microbiota; however, as an opportunistic pathogen, it could cause skin infections. Moreover, *S. aureus* could become resistant to antibiotics, and its methicillin-resistant strains are a worldwide emergency in clinical medicine. N is reported in the literature as being effective against both *P. aeruginosa* and *S. aureus*, having a MIC (minimal inhibitory concentration) of 2 μg/mL in both cases [44].

Norfloxacin loaded in the scaffolds was characterized by a microbicidal effect slightly higher against *P. aeruginosa* than against *S. aureus* and, moreover, scaffolds loaded with the free drug seem to have an antimicrobial activity higher than those loaded with norfloxacin in the nanocomposite. These could be due to the slower drug release of hybrid scaffolds compared to those loaded with norfloxacin as a free drug. However, the antimicrobial activity was sustained for 48 h.

Although a certain margin of error could be evidenced by the high variability of the results, a significant antimicrobial effect was achieved since all the scaffolds were able to decrease the bioburden by at least 100-fold (a two-log reduction). This suggests that upon implant, the scaffolds were effective for controlling and decreasing bacteria proliferation.
