Abstract
Pseudomonas aeruginosa is an opportunistic pathogen that causes hospital-acquired and chronic respiratory tract and skin infections. Treatment often fails due to antibiotic resistance. Challenges in treating antibiotic resistant P. aeruginosa infections have sparked interest in alternative therapies such as antimicrobial peptides (AMPs). AMPs target multiple cellular processes, and bacteria are less likely to develop resistance compared to traditional antibiotics. The synthetic cationic peptide DJK-5 has potent activity against P. aeruginosa and synergises with conventional antibiotics such as ciprofloxacin. DJK-5 targets the bacterial stringent stress response by binding and degrading the signalling molecule (p)ppGpp. Although DJK-5 is a promising candidate for the treatment of P. aeruginosa infections, the evolution of resistance to DJK-5 has not been investigated previously. To assess the development of resistance in P. aeruginosa PA14, experimental evolution was carried out with increasing concentrations of ciprofloxacin, DJK-5, and ciprofloxacin with 1 μg/mL DJK-5 added, under both static and shaking incubation conditions. Minimum inhibitory concentration (MIC) determination was carried out for all mutants after experimental evolution. After 30 passages (one per day), the MIC of DJK-5 against DJK-5 evolved mutants increased by 2- to 8-fold relative to the wild-type (15.63 μg/mL), with no difference between static or shaking incubation conditions. In contrast, after 14 passages ciprofloxacin-evolved mutants had a 50- to 1000-fold increase from the ciprofloxacin MIC of wild-type P. aeruginosa (0.125 μg/mL), particularly under static conditions. Mutants evolved to the combination of ciprofloxacin and DJK-5 showed similar ciprofloxacin resistance as ciprofloxacin alone, while the MIC of DJK-5 remained unchanged. Hypermutable strains were identified in DJK-5 and combination evolved populations, which have increased mutation rates and therefore had increased resistance to DJK-5, and ciprofloxacin compared to other mutants isolated from the same populations. Whole genome sequencing was carried out to identify mutations conferring ciprofloxacin and DJK-5 resistance. Unexpectedly, DJK-5 evolved mutants were identified as the strain P. aeruginosa PAO1, whereas the ciprofloxacin-evolved and combination-evolved mutants were identified as P. aeruginosa PA14 through whole genome sequencing. The source of this contamination could not be determined. Mutations identified in the DJK-5 evolved mutants included the two-component system gene pmrB, which confers resistance to cationic peptides. Ciprofloxacin-evolved and combination-evolved mutants carried known mutations associated with ciprofloxacin resistance in the genes gyrA, gyrB, parE, and mexS. My results showed that P. aeruginosa was able to develop resistance to DJK-5, but DJK-5 was unable to attenuate the evolution of ciprofloxacin resistance. This project provides insight into the mechanisms underlying DJK-5 resistance, and the potential of implementing DJK-5 as a treatment for P. aeruginosa infections.