Abstract
BACKGROUND: P. aeruginosa (PA) causes severe infections in immunocompromised patients and is increasingly antibiotic resistant. In addition, the formation of biofilm by PA makes effective treatment difficult. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are novel antisense antimicrobials that are designed to target pathogens in a gene-specific way and prevent translation of protein. We have demonstrated in vitro and in vivo activity of lead PPMOs in PA; however, it remains unclear whether PPMOs remain active in the biofilm setting over time and if synergy can be achieved with traditional antibiotics. METHODS: P. aeruginosa (GFP-PAO1) was dosed with a rhodamine-labelled PPMO targeting acpP. Samples were fixed at different time intervals and imaged by fluorescent microscopy. A Pearson’s coefficient was recorded for each dose and time condition. Minimum biofilm eradication concentration plates were used to treat PA biofilm with acpP or rpsJ targeted PPMOs in daily dosing experiments. Fixed synergy assays were performed on mature biofilm via 24-hour Q8 dosing with PPMO, antibiotics or control. Biofilm burden was quantified by CFU enumeration. RESULTS: PPMOs co-localized with PA in a dose- and time-dependent fashion. Pearson coefficients increased from 0.0583 at the 30-minutes to 0.4118 and 0.4976 at the 3- and 5-hour time points respectively. In addition, biofilm reduction was dose-dependent. In daily dosing experiments, the 3’RXR4-AcpP PPMO resulted in a 5-log reduction in biofilm burden compared to control on day 7 [p< 0.0001]; the 5’RXR4-RpsJ PPMO resulted in a 5.5-log reduction in biofilm burden compared to no treatment control [p< 0.0001]. PPMOs incubated with tobramycin or aztreonam were found to have synergistic (FIC< 0.5) or antagonistic effects (FIC > 1) in biofilm and planktonic killing depending on the combination tested. CONCLUSION: Lead PA PPMOs demonstrate dose and time-dependent biofilm eradication over multiple days of therapy. In addition, some PPMOs demonstrate synergy with other small molecule antibiotics that are used to treat certain patients with PA infections. PA PPMOs could be an innovative treatment modality for infection due to Pseudomonas. DISCLOSURES: David E. Greenberg, MD, University of Texas Southwestern Medical Center: Dr. Greenberg has numerous patents on PPMOs