Peptide-Induced Microbial Cell Lysis Assay

The determination of 260 nm absorbing material leakage from microbial cells induced by the peptides was based on a method described by Carson et al. [92 (link)] and Yasir et al. [93 (link)]. Microbial cell suspensions were prepared as described above, in Section 4.6.1. A total of 200 μL of these microbial suspensions were added to PCR grade DNA LoBind^® 1.5 mL tubes (Eppendorf, Hamburg, Germany), containing 200 μL of peptides, diluted in sterile saline solution. Final inocula were of 1 × 10⁸ CFU/mL and MICs and MBCs or MFCs (or 2x MICs when peptides had no bactericidal or fungicidal activity) were tested for peptide analogs 1, 5, 6, 7, and 8. 50 μL of these mixtures were withdrawn immediately after the addition of the cell suspensions to the peptide solutions (T0), diluted in saline solution (1:10), and filtered through 0.22 μM cellulose acetate filters. A total of 200 μL of these filtrates were then added in UV-STAR^® UV-transparent microplates (Greiner Bio-One, Frickenhausen, Germany). The same procedure was done after an incubation time of 120 min at room temperature. The 260 nm absorbance values were measured with a BioTek Synergy H1 spectrophotometer (Agilent, Santa Clara, CA, USA). Three independent repetitions, including three technical replicates, were conducted for each microorganism and peptide concentration.