3.8. Microbiological Analysis

The minimum bactericidal concentration (MBC) of the antimicrobial compounds was obtained using the macrodilution method [62]. The MBC was defined as the lowest concentration of the antimicrobial compound that would kill 99.9% of E. coli cells. The antimicrobial assay was performed in a liquid medium to allow the contact between bacteria and the free or encapsulated antimicrobials during incubation, followed by plating to enumerate the remaining viable colony-forming units (CFUs). Different compound concentrations were prepared in Erlenmeyer flasks with 15 mL of TSB for each free EOC in accordance with previous studies considering the obtained MBC values in those works [52,53]. The tested concentrations were 50, 100, 150, 200 and 250 μg/mL for Thy and Car and 125, 250, 500, 750 and 1000 μg/mL for Cin. Then, Erlenmeyer flasks, including the controls containing only the TSB medium, were inoculated with 10 µL of the E. coli suspension to obtain a final concentration of 10⁶ cells/mL. Finally, all the flasks were incubated with orbital stirring (150 rpm) at 37 °C for 24 h. In order to enumerate the viable cells after the antimicrobial treatment, decimal serial dilutions were prepared in sterile distilled water and 100-µL aliquots thereof were spread on Tryptone Bile X-glucuronide (TBX) plates. After plate incubation at 37 °C for 24 h, the colonies were counted, and the obtained results were expressed as log CFU/mL. To perform the M41–EOC–Z antimicrobial assays, the synthesized particles were added directly to the TSB contained in the Erlenmeyer flasks to obtain suspensions of each M41–EOC–Z microdevice at concentrations of 0, 1, 2, 4, 8 and 12 mg/mL, and the subsequent procedure was the same as that followed for free antimicrobials (vide supra). Positive controls, where the E. coli inoculum was allowed to grow without alterations, and negative controls, where the non-treating system M41–Z was added at the maximum particle concentration (12 mg/mL), were also performed. All the treatments (which include controls, free EOCs and M41–EOC–Z microdevices) were tested in triplicate.

To develop this experiment, suspensions of bacteria-inoculated TSB with a final concentration of 10⁶ cells/mL were prepared and incubated under different conditions for 5 h. The studied conditions included bacteria treated with free Cin (400 μg/mL) or M41–Cin–Z (4 mg/mL) and also positive and negative controls (non-treated bacteria or bacteria treated with 4 mg/mL M41–Z, respectively). Treatment concentrations were chosen based on the microbial count results (see Section 3.8.1) with the aim of testing concentrations where both live and dead cells could be visualized in the same sample. The cells in each suspension were stained with a two-color fluorescent kit, LIVE/DEAD^® BacLight™ (Invitrogen, ThermoFisher Scientific, Renfrew, UK), used for visualizing the viable and remaining dead bacteria according to membrane cell integrity. The two-color kit is composed of SYTO 9 (green fluorescent nucleic acid stain) that labels all the microbial cells with either intact or damaged membranes and propidium iodide (red fluorescent nucleic acid stain) that passes only across damaged membranes, so it labels only the dead cells that remain present in the media. Propidium iodide reduces SYTO 9 fluorescence when both dyes coexist in the same cell, resulting in red labelling. This results in a green/red labeling of the living/dead cells, respectively. For that, 0.8 μL of the aforementioned kit (SYTO 9 and propidium iodide mixed at a ratio 1:1) were added to 500 μL of each studied suspension. The mixture was incubated in the dark for 10 min to allow the penetration of dyes. Then, 5 μL of stained cells were placed over poly-L-lysine-covered slides (Sigma-Aldrich, Madrid, Spain) and sealed with a coverslip. The samples were finally observed under a Motic BA310E trinocular microscope equipped with an Epi-Led module with a 3W Led 470 nm illuminator bulb, MB barrier filter (AT480/30×, AT505DC and AT515LP fluorescence filters) and a Moticam 3+ camera. The obtained fluorescence images were acquired on a dark field with a 100× objective and have a pixel size of 0.156 µm (64 px = 10 µm).