Abstract
<jats:title>ABSTRACT</jats:title> <jats:p> <jats:italic>Pseudomonas aeruginosa</jats:italic> remains one of the most important clinical pathogens for which new drugs are needed, due to its resistance machinery. Consequently, there is an increasing effort to develop new and effective treatments against this pathogen. We recently showed that tetrasodium ethylenediaminetetraacetic acid (tEDTA) exhibits promising antibacterial and antibiofilm activity against <jats:italic>P. aeruginosa</jats:italic> in advanced biofilm models. tEDTA is known to chelate divalent cations, with predicted effects on the outer membrane; however, a full understanding of how this kills <jats:italic>P. aeruginosa</jats:italic> is lacking. Also, it is currently not clear how slowly or rapidly <jats:italic>P. aeruginosa</jats:italic> will evolve resistance to this treatment. Using membrane disruption assays and RNA-seq, we showed that tEDTA disrupts bacterial membrane potential and permeabilises <jats:italic>P. aeruginosa</jats:italic> membranes. RNA-seq revealed the significant upregulation of genes involved in the transport of iron, phosphate, potassium, and magnesium ion. The <jats:italic>arnABCD</jats:italic> operon which is involved in lipid A biosynthesis was also upregulated. Using a 7-day evolutionary ramp approach, we showed that <jats:italic>P. aeruginosa</jats:italic> could not evolve resistance to tEDTA under strong selection. Lastly, we carried out a cytotoxicity assay with Human Epithelial type 2 (HEp-2) cells and showed that there was reduced cytotoxicity of tEDTA compared to meropenem. This study provides good insight into the mechanism of action of tEDTA and further evidence of its potential as an alternative to antibiotics for <jats:italic>P. aeruginosa</jats:italic> infections. </jats:p>