*3.3.3 Fe2O3 nanoparticles*

Iron oxide (III) is a very stable oxide, it crystallizes in hexagonal form and is found in nature as the mineral hematite α-Fe2O3. The nanostructures of this oxide take different forms as they are nanowires, nanotubes, nanospheres, etc. [47]. Although its synthesis has been widely studied, its possible antibacterial effect not. The Fe2O3 NPs bactericidal effect against *E. coli* and *S. aureus* has been reported, where an increase of this effect is observed, as the concentration of iron oxide NPs increases [48]. A bactericidal effect has also been seen on *P. aeruginosa* with a minimum inhibitory concentration of 0.06 mg/L [49]. Another study reports on the bactericidal activity of nanostructured hematite against a variety of Grampositive and Gram-negative bacteria; *P. aeruginosa*, *S. aureus*, *K. pneumoniae*, *Lysinibacillus sphaericus* and *Bacillus safensis* [50]; proposing some mechanisms of action depending on the activity observed in each stage of the growth of the bacteria in question. A bactericidal effect of NPs of Fe2O3 against *S. epidermidis* has even been determined [51].

From its properties, its possible application in the remediation of the environment and water, as well as in the biomedical area, has been proposed, due to the different studies of cytotoxicity that have been carried out [47].

#### *3.3.4 MgO nanoparticles*

Magnesium oxide is in nature as the mineral periclase [52]. The antibacterial activity of MgO against Gram-positive and Gram-negative bacteria has been reported. It has been proposed that MgO NPs can damage the cell membrane causing the loss of intracellular contents and causing the death of bacterial cells [53]. The generation of reactive oxygen species has been attributed to the surface alkalinity of the MgO NPs [54]. The antibacterial activity of NPs of MgO against Gram-negative bacteria has been evaluated; *E. coli* and *P. aeruginosa* (500 and 1000 μg/mL) and in a Gram-positive bacterium; *S. aureus* (1000 μg/mL) [55]. The MgO NPs potentiated lipid peroxidation induced by ultrasound in the liposomal membrane. In this case the mechanism of action could be associated to the presence of defects, or to the lack of oxygen on the surface of the NP, leading to lipid peroxidation and the generation of reactive oxygen species [55]. The antibacterial effect and mechanism of action of NPs of MgO against strains of *Campylobacter jejuni*, *E. coli* and *Salmonella enteritidis* has been studied [56]. In this case, it was observed that the permeability of the bacteria's membrane, after exposure to the MgO NPs, was compromised, finding the presence of hydrogen peroxide that would subsequently cause cell death. Studies of *P. aeruginosa* and *S. aureus* versus MgO NPs showed a greater zone of inhibition in *S. aureus* than in *P. aeruginosa* [56]. Based on previous work, the authors note that the bactericidal action of MgO NPs may be due to the binding of surface oxygen to bacteria. As the surface area of the particles increases, the concentration of oxygen ions on the surface increases, which results in a more effective destruction of the cytoplasmic membrane and the cell wall of the bacteria.

**21**

*In vitro Antimicrobial Activity Evaluation of Metal Oxide Nanoparticles*

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

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

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

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

Regulatory agencies require adherence to well established evaluation systems. Regulatory tests applicable to disinfectants, antiseptics or therapeutic antimicrobials vary greatly; and could include to nanoparticles with potential use as

When evaluating chemical disinfectants, bacterial endospores are considered the microbial life-form hardest to kill, followed in descendent order by mycobacte-

In the United States, the Food and Drug Administration (FDA) regulates chemical sterilants and high level disinfectants (HLD) that are used to reprocess medical instruments [59]. The AOAC International sporicidal and tuberculocidal tests are the accepted methods for evaluation. For a liquid chemical sterilant, the FDA standard tolerates no failures in the AOAC sporicidal test 966.04, and accepts no survivors in simulated-use testing with a challenge inoculum of six logs of spores. The FDA defines HLD as sterilants used under the same contact conditions but for only the contact time needed to reduce *Mycobacterium bovis* in 6 log10 in the tuberculocidal test 965.12 [60]. Moreover, to be approved, the disinfectants should be subjected to worse case scenarios, such as the presence of organic or inorganic

In Europe, the CEN/TC 216 technical committee produces current and future disinfectant testing standards [61]. Standard EN-14885-2006 indicates test methods to be used to substantiate claims for products intended for instrument disinfection [62], including mycobacterial/tuberculocidal (EN-14348, EN 14563), bactericidal (EN-13727, EN-14561) and fungicidal (EN-13624, EN-14562) activity tests but the terms "sterility, sterile, sterilization, sterilant" fall outside the scope of CEN/TC 216. In the US, high, intermediate and low level disinfectants, are regulated by the Environment Protection Agency (except HLD intended to reprocess medical instruments, which fall under FDA's jurisdiction). Intermediate level

microbial species at the time, and rarely against multi-species biofilms.

*DOI: http://dx.doi.org/10.5772/intechopen.84369*

**metal oxide**

real-life scenarios.

**4.1 Regulatory testing**

antimicrobials.

*4.1.1 Disinfectants*

ria, bacteria in vegetative form, and viruses.

contamination, and under simulated use conditions.

new antimicrobials [57, 58].
