Experimental evolution to identify undescribed mechanisms of resistance to a novel cationic peptide antibiotic

Introduction: A key strategy for resolving the antibiotic resistance crisis is the development of new drugs with antimicrobial properties. The engineered cationic antimicrobial peptide WLBU2 (also known as PLG206) is a promising compound that can clear Gram-negative and Gram-positive populations and infections even when bacteria are embedded in biofilms. Despite several attempts, no definitive mechanisms of resistance to WLBU2 had been found. Here, we used experimental evolution followed by whole genome sequencing to detect novel genetic pathways and probable mechanisms of resistance to this peptide. Methods and Results: We propagated populations of wild-type and mutator Pseudomonas aeruginosa in the presence of WLBU2 and performed whole genome sequencing (WGS) of evolved populations. Populations that survived WLBU2 treatment acquired a minimum of two mutations, making the acquisition of resistance more difficult than for most antibiotics, which can be tolerated by mutation of a single target. Major targets included the orfN and pmrB genes, previously described to confer resistance to other cationic peptides. More surprisingly, mutations that increase aggregation such as the wsp pathway were also selected despite the ability of WLBU2 to kill cells growing in a biofilm. Conclusion: Experimental evolution and WGS identifies genetic targets and actions of new antimicrobial compounds and can predict pathways to resistance to new antibiotics in clinical practice.