Antibiotic potentiators and delivery to persisters

Abstract

Bacterial persisters are cells that transiently withstand the lethal effects of antibiotic therapy without acquiring genetic mutations. It is increasingly clear that the presence of persisters accounts for the recalcitrance and relapses of chronic bacterial infections and has also been associated with the evolution of bacterial resistance. Sensitization of these semi-dormant persisters through metabolite-supplementation has been shown to be effective against persisters of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. However, this metabolite-aminoglycoside adjuvant therapy needs a suitable vehicle to reach the site of infection for clinical applications. Here, we introduce the lipid-polymer hybrid nanoparticle (LPN) system, which is a non-toxic delivery system proven to be capable of achieving localized and sustained release of antibiotics to bacterial biofilms. Co-delivery of gentamicin and glucose using LPN showed up to a five-order decrease in the viability of persisters compared to the free formulation and full eradication of persisters was achieved using the LPN system in both 4 h and 24 h of treatment. However, supplementation of the metabolites alone was not effective against persisters when bacteria were treated directly in the spent growth medium. We successfully potentiated gentamicin in such situations through the addition of alkaline buffering agents. We hypothesized that the addition of alkaline buffering agents helped to elevate and maintain the local pH thus restored the lethality of gentamicin. Supplementation of alkaline buffering agents alone showed a two-order decrease in the viability of persisters and synergy was found when metabolites and buffering agents were added together, showing a full eradication at a gentamicin concentration of 100 μg/ml. Furthermore, we explored the possibility of using oxygen as an adjuvant. Overall, these results suggest specific metabolites, alkaline agents and adequate oxygen are essential components to potentiate aminoglycosides and a proper encapsulation ratio is crucial to achieving clinical success against S. aureus persisters.