Multidrug Resistant Pseudomonas aeruginosa: Targeting essential genes as a novel therapy for healthcare-associated pneumonia

Julie England

Introduction: Pseudomonas aeruginosa is a major cause of healthcare- and ventilator-associated pneumonia (HAP, VAP). This complication produces significant increases in mortality, morbidity, and cost of care.1,2 20% of Pseudomonas pneumonia cases are caused by multidrug resistant (MDR) strains.3 Importantly, the presence of an MDR strain is the most important predictor of inadequate initial antibiotic therapy (IIAT).4 IIAT is an independent risk factor for mortality in the ICU.4,5 Traditional antibiotics have become increasingly ineffective against MDR P. aeruginosa HAP/VAP, and we are in desperate need of a reliably effective therapy. One potential therapy involves the silencing of bacterial essential genes. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are capable of entering a bacterium, binding the mRNA of the target gene, and blocking its translation.6,7 The loss of an essential protein product results in death of the bacterium. The purpose of this investigation is to identify PPMOs capable of anti-pseudomonal activity, and to clarify their potential for translation into clinical therapies.  Methods: To identify effective carrier peptides, LacZ expression was induced in P. aeruginosa. Strains were incubated with anti-LacZ and scrambled PMOs, conjugated to peptide 1, KFFKFFKFFK, or peptide 2, (RXR)­­4. LacZ activity was measured.6 To quantify the in vitro efficacy of the acpP-(RXR)­­4 PPMO, an effective PMO sequence against acpP mRNA was identified, conjugated with the (RXR)­­4 carrier peptide, and incubated in P. aeruginosa culture at increasing doses.7 To investigate the activity of PPMOs against P. aeruginosa pneumonia in vivo, mice were inoculated with P. aeruginosa, incubated 6 hours, treated with anti-acpP PPMOs, saline, or scrambled PPMO, and then sacrificed at 24 hours.  Results: Maekawa, et al. identified (RXR)4 as an effective carrier peptide against Pseudomonas without off-target toxicities.6 Ghosal, et al. determined that an effective anti-acpP PMO was 11 bp long, from -6 bp to +5 bp, across the translation start codon.7 This acpP-(RXR)­­4 PPMO demonstrated activity against P. aeruginosa in vitro.7 In the mouse model of acute pneumonia, Howard, et al. demonstrated that the acpP-(RXR)­­4 PPMO reduced CFU in the lungs by almost 3 logs. Histologically, acpP-(RXR)­­4 PPMO-treated lungs showed reduced inflammatory infiltrate.Conclusions: acpP-(RXR)­­4 PPMOs effectively inhibit growth of P. aeruginosa in vitro and in vivo, and successfully treat P. aeruginosa pneumonia in a murine model. Given these favorable results, and the recent FDA approval of the PMO drug Eteplirsen10 for muscular dystrophy, PPMOs are a promising option for future clinical studies in Pseudomonas pneumonia.

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