Elucidating bacterial responses to the novel antimicrobial AGXX

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SUMMARY New antimicrobial strategies are badly needed, as the spread of antibiotic resistance is rapidly compromising the effectiveness of current antibiotics. Moreover, new drug development has not kept pace with the rise of drug-resistant pathogens. Pseudomonas aeruginosa is particularly difficult to treat and commonly found in the lungs of cystic fibrosis patients or in patients with burn wounds. Due to the emerging antibiotic crisis, efforts are now focused on finding alternative treatment strategies. Silver-containing compounds represent such opportunity due to their multi-specific ability to inhibit bacterial growth. One example is silver sulfadiazine, a topical formulation that is often administered to treat or prevent acute P. aeruginosa wound infections. The novel silver containing surface coating AGXX was recently developed as a promising compound with antimicrobial properties. Composed of the two transition metals silver and ruthenium which form a micro-galvanic cell, AGXX kills gram-positive bacteria through the formation of reactive oxygen species, such as hydrogen peroxide. However, its effect on gram-negative bacteria such as P. aeruginosa as well as their responses to AGXX exposure have not yet been studied. We found that AGXX elicits strong proteotoxic effects in P. aeruginosa even at sublethal concentrations, and that the compound is significantly more potent than silver sulfadiazine, the gold standard for the treatment of burn wounds. Moreover, we discovered that the bactericidal activity of sublethal concentrations of AGXX is up to 50,000-fold higher in the presence of sublethal aminoglycosides concentrations, indicating a potential application for AGXX as an adjuvant. We will now investigate the molecular mechanism behind the synergy of aminoglycoside antibiotics and AGXX. In Aim 1, we will test our hypothesis that AGXX-induced ROS production disrupts the cellular iron-sulfur cluster pool and therefore triggers hydroxyl radical production via the Fenton reaction. As a result, we expect to observe increased membrane disruption, potentially facilitating an elevated aminoglycoside influx into the cell, which is responsible for increased bacterial killing that is observed after simultaneous treatment of P. aeruginosa with AGXX and aminoglycosides. Furthermore, we will determine the effect a combinational treatment has on P. aeruginosa biofilms and persister cells and on isolates of other clinically relevant bacterial species. In Aim 2, we will use independent unbiased and targeted approaches, including Tnseq and subsequent phenotypic characterization of transposon mutants, to identify and determine P. aeruginosa-specific responses to and defenses against AGXX treatment. Moreover, we will assess the cytotoxicity of this compound in different cell lines of relevance. These findings will guide efforts to devise strategies to implement AGXX as a potential antimicrobial and adjuvant in eradicating P. aeruginosa infections.