Mechanistic insights in the antibiotic tolerance of Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa is a pathogen of high clinical relevance, responsible for many deadly infections and showing high resistance to current antimicrobial agents. In the human body, P. aeruginosa grows as a microbial community embedded in a matrix of extracellular polymeric substances, called biofilm, which promotes the survival of bacteria upon antibiotic exposure and environmental stresses. The goal of this thesis is to identify genetic determinants responsible for P. aeruginosa biofilm tolerance to antibiotics that may serve to identify new targets for drug development and innovative therapeutic strategies. To do so, we selected biofilm relevant genes based on previous transcriptomic studies and characterized their involvement in biofilm tolerance to antibiotics using the public P. aeruginosa MPAO1 transposon insertion library. We showed that the inactivation of the Pf phage gene, PA0720, or the flagellin gene, fliC, led to the complete removal of biofilms with 4-fold lower gentamicin concentration. We demonstrated that the downregulation of certain genes (e.g., coxC, pprB, PA3785) correlates with enhanced gentamicin tolerance in P. aeruginosa. Finally, we highlighted that the repression of the flagellum gene, flgE, in cystic fibrosis infections is an active mechanism of adaptation employed by P. aeruginosa to alter its biofilm structure and promote antibiotic tolerance. In conclusion, this thesis gives insight in what should be considered when investigating P. aeruginosa clinical strains and reveals new targets for drug development and new markers to predict antibiotic resistance