Background

Functional coatings are being widely investigated owing to the valuable advantages they offer to the area of materials engineering. They are capable of introducing new surface properties, with no changes in the material bulk characteristics. Among their functionalities, antibacterial coatings have demonstrated great importance for biomedical applications (Mateescu et al., 2015). In this sense, nanotechnology provides substantial tools for the fabrication of antibacterial coatings. Our group has achieved the synthesis of nanostructured coatings with broad spectrum bactericidal activity, without utilizing antibiotics or bacteria repelling substances (Scilletta et al., 2019). Here, only the superstructure is responsible for the bactericidal activity. To find the specific superstructure capable of bacteria killing many assays with distinct coatings configurations have been performed. All the coatings were composed of poloxamer block copolymers (also named as Pluronic^®) within a titania matrix. Thus, film coatings were prepared using two copolymers which differ in the length of the hydrophilic and hydrophobic portions (Pluronic F127 and Pluronic P123). The quantities of the copolymers varied systematically, therefore modifying the titania: pluronic mol ratios. These modifications modulate the mesophase, which we proved is directly related to the bactericidal capacity. The mesostructured titania thin film coatings were synthesized by the sol-gel method in combination with evaporation-induced self-assembly of the pluronic micelles and deposited by spin-coating on glass substrates (Scilletta et al., 2019).

In order to find the best bactericidal performance, the aforementioned distinct coating configurations were compared based on their antibacterial efficacy. We utilized three methods to determine the most effective antibacterial coating: counting of the number of colony forming units (CFU), live/dead staining, and quantification of extracellular DNA in suspension. To perform the counting of CFU after bacterial incubation on the films we modified a previous protocol by Merritt et al. (2006). Afterwards, coatings presenting the best bactericidal performance were further studied to clarify the mechanism of bacteria killing. In this regard, fluorescent staining with SYTO 9 and propidium iodide (PI) was utilized to determine the integrity of the bacteria membrane. After incubating the bacterial inoculum on the films, the recovered suspension was dyed with this mixture. PI only diffuses into damaged membrane cells and binds to DNA with high affinity, labeling those cells in red. SYTO 9 only stains live cells with intact membranes, labeling them in green. This protocol is a modification of the one published by Smith and Hunter (2008). Lastly, the presence of extracellular DNA was studied as an indicator of cell membrane disruption. To perform this determination we modified the previous protocols from Chen and Cooper (2002) and from Yuan et al. (2019).

As a result of the three considered aspects, this protocol describes an integral approach for the determination of antibacterial activity of film coatings. The protocol can be utilized to evaluate the effects of any coatings on bacterial viability, which includes bactericidal superstructures like the one studied by us, and drug-loaded coatings, either with traditional antibiotics or novel antibacterial agents.