ADP-MoA-Pa: a platform for screening antibiotic activity and their mechanism of action in Pseudomonas aeruginosa.

Abstract

Aims: The emergence of multidrug-resistant bacteria poses a significant threat to global public health. To address this crisis, there is an urgent need to identify and characterize novel antibacterial molecules. This study aimed to develop the ADP-MoA-Pa platform to facilitate the discovery of new antibiotics and provide preliminary insights into their mechanisms of action (MoA).

Methods and results: The ADP-MoA-Pa platform enables the simultaneous visualization of antibiotic activity (growth inhibition) alongside one of three classic mechanisms of action in P. aeruginosa: DNA damage/inhibition of DNA replication, protein synthesis inhibition, or cell wall damage. To construct the platform, the promoter regions of recA, ampC and armZ of P. aeruginosa PA14 strain were each amplified and fused to a promoterless luxCDABE operon in vector pUC18T-mini-Tn7T-lux-Gm. The constructs were electrotransformed into strain PA14 where they integrated in the chromosome. Each promoter fusion was activated by the expected antibiotics on plates and in liquid media, thereby demonstrating proof of concept. The armZ:: luxCDABE fusion responded to protein synthesis inhibitors such as macrolides, chloramphenicol, tetracyclines, and aminoglycosides. The ampC:: luxCDABE fusion was induced by β-lactams, while the recA:: luxCDABE fusion was activated by quinolones and nitrofurantoin. Interestingly, under some conditions, ciprofloxacin also activated ampC and armZ, though to a lesser extent.

Conclusions: The ADP-MoA-Pa platform is a low-cost, readily implementable tool with significant potential for high-throughput screening of antimicrobials. It offers a promising avenue for identifying and characterizing novel antibiotics against Pseudomonas aeruginosa and other bacterial species, contributing to the global effort to combat multidrug-resistant pathogens.