Resurrecting the Dead: Mitigating Efflux-Pump Inhibitor Toxicity using a Liposomal Delivery System to Recover Efficacy of Antimicrobial Drugs

Abstract

Introduction: Antimicrobial resistance (AMR) has become concerningly prevalent on a global scale as many infectious agents have evolved to evade antimicrobials effects, making it difficult to treat infectious diseases. Pseudomonas aeruginosa is a multiresistant bacteria that requires urgent attention as it is detrimental in lung infections. Activity of membraneembedded efflux pumps, such as the MexAB-OprM pump, is a principal mechanism by which bacterial species become resistant to antimicrobials. Efflux pump inhibitors (EPIs) have recently emerged as a strategy to prevent the expulsion of administered antimicrobials, thereby resensitizing resistant bacteria to antibiotics. Phenylalanine-arginine β-naphthylamide (PAβN) is an EPI that inhibits a number of different pumps, including the MexAB-OprM efflux system. Despite EPIs providing a partial solution to AMR, they have been shown to be toxic to humans, which has impeded their entry into clinical application. We propose that by inserting the PAβN into a liposomal delivery system, the cytotoxic effects against human cells will be lowered without decreasing the EPI's inhibitory activity. Methods: Resistant P. aeruginosa strains will be administered with liposomes jointly encased with PAβN and ampicillin to ensure both are delivered to the same cell, while testing for changes in antimicrobial efflux activity and bacterial growth, indicative of restored antimicrobial effectiveness. Efflux activity and inhibited growth will be measured using an ethidium bromide efflux assay and a minimum inhibitory concentration assay, respectively. Human pulmonary epithelial cells will be exposed to liposomal-encased PAβN to study changes to EPI cytotoxicity, with cell viability being measured using a Cell Titer Blue assay. Results: Liposomal EPI are expected to maintain inhibitory activity and resistant bacteria would become re-susceptible to antibiotics when treated with the liposomal EPI. Discussion: By analyzing efflux rate to measure the liposomal EPI's activity, its activity level should be comparable to free EPI. The resensitization assay would show that the bacteria are susceptible to antibiotics again. Conclusion: If effective, EPIs may become a potential therapeutic to combat AMR by reviving the use of antimicrobials that have become ineffective. Restoring the activity of already approved antibiotics through potential co-administration with liposome-encapsulated EPIs will be a cost-effective approach to combat AMR.