Bacterial resistance and susceptibility to antimicrobial peptides and peptidomimetics

Bacterial resistance to conventional antibiotics has become a global challenge and there is urgent need for new and alternative compounds. Antimicrobial peptides (AMPs) are under investigation as novel antibiotics. These are part of the immune defense of all living organisms; hence, they represent a valid candidate both for their antibacterial activity and for their immunomodulation features.

However, these compounds have several disadvantages once administered in vivo. These shortcomings have led to extensive attempts of improving their features with rational synthetic design. Peptidomimetics are one class of such synthetic modified peptides. The purpose of this PhD project was to determine the antibacterial spectrum and potential use of synthetic antimicrobial peptides and peptidomimetics.

Another key investigation has been the experimental development of resistance to these novel antibacterial agents. We investigated (Article 1) the antibacterial effect of selected peptidomimetics in a simulated in vivo environment using human blood plasma and serum. We speculated that the activity of peptidomimetics was hampered by the presence of blood fluids.

However, the antibacterial activity was enhanced in presence of human blood plasma but not in in presence of human blood serum. We hypothesized that complement system or clotting factors present in plasma but not in serum were causing the enhanced effect of peptidomimetics. Interestingly, in presence of heatinactivated blood matrices, the activity of the compounds decreased dramatically or no enhancement was observed, indicating that inactivation of complement has occurred.

We also determined whether the antibacterial activity of selected conventional antibiotics was affected by the presence of blood fluids and indeed the activity of a membrane active antibiotic was enhanced in presence of human plasma. We conclude that complement system and other factors present in human blood plasma interact synergistically with membrane active compounds such AMPs are.

As a result, the concentrations of peptidomimetics and peptide antibiotics needed in vivo may be lower than predicted from standard antimicrobial susceptibility testing. Unfortunately bacteria can easily adapt to AMPs in laboratory settings and we found (Manuscript 2) that in Escherichia coli through an adaptive evolution experiment.

We hypothesized that evolution of resistance to the combination would be slower than to the single compounds. However, the lineages exposed to P9-4 (alone or in combination) were the slowest adapting as compared to the other treatments. We suggest that the AMP P9-4 could be considered a promising candidate for future application in clinical settings, because of its slow resistance development rate.

Using whole-genome sequencing, we investigated the genetic basis of resistance in the adapted lineages and derived clones. Deletions in the gene encoding for the enzyme CDP-glycerophosphotransferase were the most common variants, indicating that a common sequence of mutation events has led to development of resistance.

The zeta potential of adapted lineages was less negative than that of the wild type and we therefore hypothesized that a potential mechanism of resistance relies on surface charge modifications. In Manuscript 3 we investigated the stability of the evolved resistance by re-cultivating selected resistant clones in absence of compound.

Several clones retained resistance after re-cultivation in absence of compound. Genome analyses demonstrated that deletions in the gene encoding for the enzyme CDP-glycerophosphotransferase were still present after re-cultivation. Thus, this enzyme may indeed play a key role in the mechanism of resistance.

Cross-resistance is a common feature of resistant microorganisms and we therefore determined whether the adapted, resistant clones had altered susceptibility to other antibacterial compounds. The resistant clones were also resistant to compounds with intracellular activity. However, the same clones were as susceptible as the wild type when exposed to membrane-active compounds with specific features such as lipidation, incorporation of D-amino acids and presence of IR motifs.

Thus, the concern that AMPsresistant clones may be a threat to our immunity may be overestimated. In conclusion, this PhD project supports the belief that bacteria hold the potential to develop resistance to each novel antibacterial agent. Nevertheless, strategies to circumvent resistance exist and must be pursued.