Peptidomimetics

MIC was determined by using the micro-dilution method according to guidelines of the Clinical and Laboratory Standards Institute [57] . Peptidomimetic 1 and erythromycin 1∶2 serial dilutions were prepared from 1024 µg/mL stock solutions to give a final range of 512-0.5 µg/mL in the wells. Ampicillin 1∶2 serial dilutions were prepared from a 256 µg/mL stock solution to give a final range of 128-0.13 µg/mL in the wells. Polymyxin B and gentamicin 1∶2 serial dilutions were prepared from a 64 µg/mL stock solution to give a final range of 32-0.03 µg/mL in the wells. Also, 1∶2 serial dilutions of the AMPs KR-12, IsCTp, melittin and PEP-1-K were prepared from 64 µg/mL stock solutions to give a final range of 32-0.03 µg/mL in the wells; however this was performed in 1% peptone instead of in MHB since activity was drastically diminished in the latter (not shown).
The population lineages were tested for their susceptibility towards peptidomimetic 1, polymyxin B, gentamicin and ampicillin. Single-culture isolates selected from these populations were additionally tested against peptidomimetic 1, polymyxin B, erythromycin and the five reference AMPs. To estimate the level of cross-resistance to closely related peptidomimetics, MIC determinations were performed for peptidomimetics 2 and 3 (Figure 1) towards selected isolates. The bacterial suspensions were grown overnight in MHB at 37°C for determination of the population MIC, or individual isolates were grown overnight on non-selective BHI agar at 37°C. MIC determination performed on the wild-type strain using re-suspended colonies, as well as on a culture grown overnight in broth, showed that pre-growth in broth had minimal effect on the MIC results. In order to determine the population MIC, the respective compound (peptidomimetic 1 or polymyxin B) was added to give a concentration corresponding to 32×MIC to maintain the selection pressure of the freezer-revived lineages, and then MIC determination was performed on the outgrown population (i.e. ∼10⁹ CFU/mL). In all experiments, bacterial cells were suspended in 0.9% saline to give a turbidity of 0.13 at OD₅₄₆ (approximately 1×10⁸ CFU/mL) and diluted in MHB pH 7.4 to a final concentration of 5×10⁵ CFU/mL in each well. Polypropylene plates (Nunc 442587) were used to minimize peptide binding, and the incubation time was 18–20 hours at 37°C. Incubation time was additionally extended up to 44 hours for the individual isolates due to a potential effect of growth rate on the outcome, but no effect was seen of this (not shown). MIC was found in a minimum of two technical (population MIC) or biological (isolate MIC) replicates as the lowest concentration of the AMP or antibiotic where no visible growth was observed.