Difco 0.5x nb

To examine whether the hydrogel system loaded with the peptidomimetic is able to counteract bacterial migration across surfaces, the upper chambers of a trans-well plate (Costar 3422^®, Corning Corporation, Corning, NY, USA) were coated with control saline solution, HA-BDDE hydrogel or HA-BDDE hydrogel loaded with (ri)-r(P)ApoB_S^Pro, as described by Xiaojuan Li et al. [33 (link)]. Following coating, methicillin-resistant S. aureus (MRSA WKZ-2) and E. coli ATCC 25922 bacterial cells were diluted to 4 × 10⁶ CFU/mL in Difco 0.5X NB (Becton-Dickinson, Franklin Lakes, NJ, USA), and plated into the previously coated upper chambers. The medium in the lower wells was then analyzed at defined time intervals, in order to monitor bacterial cells’ migration from the upper to the lower wells. The number of migrated bacteria was quantified by diluting and plating each sample on TSA. Following an incubation of 24 h at 37 °C, the number of colonies was counted. The experiment was performed in three independent replicates.

The antimicrobial activity of HA-BDDE hydrogel loaded with (ri)-r(P)ApoB_S^Pro was assayed on methicillin-resistant S. aureus (MRSA WKZ-2) and E. coli ATCC 25922 bacterial strains. Bacteria were grown to mid-logarithmic phase in MHB at 37 °C. Afterward, cells were diluted to 4 × 10⁶ CFU/mL in Difco 0.5X NB (Becton-Dickenson, Franklin Lakes, NJ, USA) and mixed 1:1 v/v with HA-BDDE hydrogel loaded with the peptidomimetic at a final concentration of 80 or 320 μmol L⁻¹. Following overnight incubation, each sample was diluted, plated on TSA, and incubated at 37 °C for 24 h, in order to count the number of colonies. All the experiments were carried out as triplicates.