Utilizing a novel reactive oxygen species-generating antimicrobial to potentiate antibiotics

The emerging antimicrobic resistance and decline in the discovery of new antibiotics requires drug development to consider alternative strategies. Silver-containing compounds became a promising alternative due to their multi-specific effects on inhibiting bacterial growth. We focus on the antimicrobial effects of AGXX, a novel antimicrobial composed of silver and ruthenium, which exerts its broad-spectrum effects through the production of highly cytotoxic reactive oxygen species (ROS) that inflict extensive macromolecular damage. Due to recent connections identified between ROS-stress and antibiotic activity, we hypothesized that AGXX could potentially increase the activity of conventional antibiotics. We investigated possible synergistic effects of AGXX on members of several antibiotic classes in 5 different bacterial pathogens. We discovered that combined treatment of sublethal concentrations of AGXX and aminoglycoside antibiotics exponentially enhanced their bactericidal activity and restored sensitivity in aminoglycoside resistant clinical isolates. To explore the underlying mechanism of the synergizing effects of AGXX on aminoglycosides, we utilized redox sensitive probes and found that the bactericidal effect of the combined AGXX/aminoglycosides treatment is mediated through elevated ROS production, which was attenuated with the addition of the ROS scavenger thiourea. We discovered that the increase in oxidative stress resulted in a disruption of iron homeostasis evidenced by a significant decrease in aconitase activity under combined AGXX/aminoglycoside treatment. Using spectroscopic methods and fluorescent microscopy, we further demonstrate that combined treatment of the two antimicrobials inflicted significant membrane damage, thus increasing membrane permeability. Our subsequent goal is to further delineate the mechanisms that underpin this synergistic effect by exploring how the metabolic processes and cellular targets disrupted by AGXX contributes to the synergistic effects with aminoglycosides. Our findings will potentially provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials.