Pseudomonas aeruginosa (Pae) is notorious for its high-level resistance towards clinically used antibiotics. In fact, Pae has rendered most antimicrobials ineffective, leaving polymyxins and aminoglycosides as last resort antibiotics. Although several resistance mechanisms of Pae are known towards these drugs, a profounder knowledge of hitherto unidentified factors and pathways appears crucial to develop novel strategies to increase their efficacy. Here, we have performed for the first time transcriptome analyses and ribosome profiling in parallel with strain PA14 grown in synthetic cystic fibrosis medium upon exposure to polymyxin E (colistin) and tobramycin. This approach did not only confirm known mechanisms involved in colistin and tobramycin susceptibility but revealed also as yet unknown functions/pathways. Colistin treatment resulted primarily in an anti-oxidative stress response and in the de-regulation of the MexT and AlgU regulons, whereas exposure to tobramycin led predominantly to a rewiring of the expression of multiple amino acid catabolic genes, lower tricarboxylic acid (TCA) cycle genes, type II and VI secretion system genes and genes involved in bacterial motility and attachment, which could potentially lead to a decrease in drug uptake. Moreover, we report that the adverse effects of tobramycin on translation are countered with enhanced expression of genes involved in stalled ribosome rescue, tRNA methylation and type II toxin-antitoxin (TA) systems.

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