Novel Adjuvant Approaches for Disarming Antibiotic Resistance in Clinically Relevant Bacterial Pathogens

As a response to the rising emergence of antibiotic resistant bacteria, President Barack H. Obama administered a National Action Plan in 2015 as incentive to raise awareness and re-invest in antibiotic development and alternative methods to fight bacterial infections. This $1.2 billion Action Plan set into motion five main objectives which involved extensive collaboration between U.S. and foreign governments. These objectives included: 1) slowing the spread of resistant bacteria, 2) strengthening National One-Health Surveillance efforts, 3) advancing development and rapid use of innovative diagnostic tests for characterization and identification of resistance bacteria, 4) accelerating applied and basic research and development (R&D) for novel vaccines, antibiotics, and other therapeutics, and 5) improving international collaboration on prevention, surveillance, control, and R&D efforts involved with antibiotic resistance. The work in this document describes multiple projects that focus on using antibiotic adjuvants to sensitize Gram-negative pathogens to Gram-positive selective antibiotics. In depth discussions detail the initial hits and structure-activity relationship (SAR) studies that led us to finding lead adjuvants for potentiating macrolide antibiotics against clinically relevant Gram-negative pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii. Gram-negative bacteria are more robust than Gram-positive bacteria, since they have an outer membrane that provides extra protection against antibiotic permeability. We identified a class of adjuvants that dramatically enhance sensitivity of P. aeruginosa to the macrolide antibiotic azithromycin, with reductions in the minimal inhibitory concentrations (MIC) as high as 1024-fold and activity observed across a variety of clinical isolates from cystic fibrosis patients. Furthermore, an in vivo model using the wax worm Galleria mellonella demonstrated the ability to combat P. aeruginosa when administered a combination of azithromycin (50 mg/kg) and adjuvant (50 mg/kg). Another project identified several potent adjuvants with diverse chemical scaffolds that potentiated macrolide antibiotics, specifically clarithromycin, against A. baumannii with reductions in MICs between 128- and 1024-fold, and activity observed across a variety of clinical isolates. Lastly, this document describes collaborative efforts towards synthesizing lead adjuvants that potentiate colistin, the last-resort antibiotic prescribed for multi-drug resistant Gram-negative infections. Lead adjuvants were able to sensitize chromosomally colistin resistant strains of A. baumannii substantially at low concentrations.