**4.** *In vitro* **methods for antimicrobial evaluation of nanoparticles based metal oxide**

Bacteria exposed to antimicrobials are under selective pressure to evolve and adapt, this natural process leads to antimicrobial resistance. Human kind is facing the growing threat of rapid evolution and dissemination of bacteria resistant to multiple antibiotics. There is, therefore, an urgent need to develop new antimicrobials [57, 58].

Antimicrobial agents include disinfectants, antiseptics, and antibiotics. New agents must be exhaustively tested for efficacy and safety. Evidence-based selection of the microorganisms and the evaluation system is of paramount importance for adequate interpretation of the test results, and for extrapolating from *in vitro* to real-life scenarios.

The use of nanoparticles especially based in metal oxides emerge as new antimicrobial agents, therefore it is necessary to test the efficacy of nano-antimicrobials against representative bacterial species. One known limitation of the testing systems currently in use, is that formulations are often challenged *in vitro* with one microbial species at the time, and rarely against multi-species biofilms.
